Note: Descriptions are shown in the official language in which they were submitted.
11111 , -
,
\ -
'
' RECOMBINANT DNA MOLECULES
AND THEIR METHOD OF PRODUCTION
TECHNICAL FIELD OF INVENTION
This invention relates to recombinant DNA
molecules and their method of production. This invention
relates more particularly to recombinant DNA molecules
expressed in appropriate host organisms. The recombinant
DNA molecules disclosed herein are characterized by the DNA
that codes for polypeptides with the specificity of
hepatitis B viral antigens. As will be appreciated from
the disclosure to follow, the recombinant DNA molecules may
(
be used in the detection of hepatitis B virus in man and
the stimulation of antibodies in humans against this virus:
BACKGROUND ART
,
The virus that causes hepatitis B or serum
- - hepatitis appears to infect only man. Hepatitis B virus
("HBV") infection in humans is widespread. In the United
Kingdom, United States, and Western Europe, approximately
0.1% of all blood donors are chronic carriers of HBV.
While the death rate due to viral hepatitis is not high (in
1975 in the United Kingdom it was 3 per million), there are
indications that as many as 5% of the population in the
United Kingdom and 15% of the population in the United
States have been infected. In many African and Asian
countries, up to twenty percent of the population are
chronic carriers of HBV, and over fifty percent of all
adults in those countries have been or are infected with
HBV.
;
,,...
CA 1341644 1979-12-21
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.
F 1111
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The hepatitis infection is transmitted by three
general mechanisms: (1) by parenteral inoculation of
infected blood or body fluids, either in large amounts as
in blood transfusions or in minute amounts as through an
accidental skinprick; (2) by close family or ---sexual
contact; and (3) by some mothers, who infected during
pregnancy, transmit the virus to their new-born children.
Under natural conditions, HBV is not highly contagious.
Transmission by inhalation occurs rarely, if ever.
Most HBV infections are subclinical. The
clinical illness usually lasts three to six weeks and
ranges in severity from mild to acute fulminating hepatitis
followed by cirrhosis or death. Recovery from clinical and
subclinical HBV infections is usually complete. However,
serious long term consequences result in some cases:
(1) approximately five percent of acute infections lead to
chronic carriage of hepatitis B virus antigen with its
continuing potential for infectivity to others and ongoing
liver damage; and (2) it is likely that past infection with
HBV may be wholly or partly responsible for the initiation
of a significant proportion of HBV-seronegative cases of
chronic active hepatitis, cirrhosis and primary liver
carcinoma.
Recent advances in molecular biology have made it
possible to introduce the DNA coding for specific non-bac-
terial eukaryotic proteins into bacterial cells. In
general, with DNA other than that prepared via chemical
synthesis, the construction of the recombinant DNA
molecules comprises the steps of producing a single-
stranded DNA copy (cDNA) of a purified messenger (mRNA)
template for the desired protein; converting the cDNA to
double-stranded DNA; linking the DNA 'to an appropriate site
in an appropriate cloning vehicle and transforming an
appropriate host with that recombinant DNA molecule. Such
transformation permits the host to produce the desired
protein. in addition, at least in the case of ovalbumin
DNA, it is known that appropriate fusion of the particular
CA 1341644 1979-12-21
-3-
ti
DNA to a strong bacterial promoter or expression control
sequence produces larger amounts of the desired ovalbumin
protein, i.e., about 0.5 to 1% of the total protein mass of
an E. coil cell (0. Mercereau-Puijalon et al., "Synthesis
Of An Ovalbumin-Like Protein By Escherichia COli K12 Har-
boring A Recombinant Plasmid", Nature, 275, pp. 505-510
(1978) and T.H. Fraser and B.J. Bruce, "Chicken Ovalbumin
Is Synthesized And Secreted By Escherichia coil", Proc.
Natl. Acad. Sci. USA, 75, pp. 5936-5940 (1978)).
Several non-bacterial proteins and genes have
been synthesized using recombinant DNA technology. These
include a protein displaying rat proinsulin antigenic
determinants (L. Villa-Komaroff et al., "A Bacterial Clone
= 'Synthesizing Proinsulin", Proc. Natl. Acad. Sci. USA, 75,
pp. 3727-3731 (1978)), rat growth hormone (P.H. Seeburg et
al., "Synthesis Of Growth Hormone By Bacteria", Nature,
276, pp. 795-798 (1978)), mouse dihydrofolate reductase
(A.C.Y. Chang et al., "Phenotypic Expression in E. coil Of
A DNA Sequence Coding For Mouse Dihydrofolate Reductase",
= 20 Nature, 275, pp. 617-624 (1978)), somatostatin (K.
Itakura
et al., "Expression In Escherichia coli Of A Chemically
Synthesized Gene For The Hormone Somatostatin", Science,
198, 1056-1063 (1977), United Kingdom patent specifications
2,007,675A, 2,007,676A and 2,008,123A and cognate applica-
tions in other countries), and the A and B polypeptide
chains of human insulin (D.V. Goeddel et al., "Expression
In Escherichia coil Of Chemically Synthesized Genes For
Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-110
(1979) and the United Kingdom and related patent specifi-
cations, supra).
None of the foregoing, however, is directed, as
is this invention, toward the recombinant DNA synthesis of
viral proteins such as hepatitis B virus antigens.
It is known that HBV infections cause development
of antibodies to the antigens of the virus. These anti-
bodies are the body's defense mechanism to the HBV infec-
tion. The development of such antibodies prior to exposure
CA 1341644 1979-12-21
111 -4-
or rapidly in the case of potential exposure should substan-
tially reduce and combat virus growth and spread in the
patient.
A problem however in the artificially stimulated
development of antibodies to the antigens of hepatitis B
virus is the limited host range of the virus. For example,
although highly infectious to man, experimental infection
with hepatitis B virus has been achieved in only a few
additional primates. And, the virus has not been propa-
gated in tissue culture. This limited host range and
inability to infect tissue culture cells has severely
hampered both the characterization of the virus and the
pathology of its infection and the development of rapid
=detection means and effective means of infection control
and prevention.
While there have been some attempts to employ
authentic HBV viral antigens, isolated from victims of the
HBV infection, for the development of antibodies and detec-
tion of infection, these treatments are not generally
available because of the limited supply of the active
species. Furthermore, the use of human sources for these
antigens is disfavored because of the well recognized
contamination problems in using human isolates.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred
to by providing in accordance with the invention a recombin-
ant DNA molecule characterized by a structural gene coding
for a polypeptide displaying HBV antigenicity.
By virtue of our invention, it is possible to
obtain HBV antigens and genes in substantial and uncon-
taminated quantities for vaccine preparations and for use
in detection of the viral infection and determination of
its pathology and molecular biology. Such supplies have
not hitherto been available, because of the narrow host
range of the virus and its inability to be grown in tissue
culture. .
CA 1341644 1979-12-21
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' 1111
...5_
As will be appreciated from the disclosure to
follow, the recombinant DNA molecule of the invention is
capable of producing, in an appropriate host, at least one
viral polypeptide displaying HBV antigenicity and the
structural genes that code therefor. These recombinant DNA
molecules and hosts may be utilized to prepare polypeptides
displaying HBV antigenicity and structural genes coding for
these polypeptides. These products may also be identified
and characterized and are useful either as produced in the
host or after appropriate derivatization or modification in
compositions and methods for improving production of these
products themselves and for detecting HBV infection, tracing
its pathology and stimulating the production of HBV anti-
bodies in humans.
In accordance with the invention we also provide
a process for producing a recombinant DNA molecule, charac-
terized by linking a DNA sequence prepared from the endog-
enous DNA of a_Dane particle to another DNA sequence pre-
pared from a source other than the Dane particle.
= 20 Our process may be distinguished from the prior
processes above mentioned in that none of the prior pro-
cesses employs a natural gene or DNA for a particular
protein for construction of the recombinant DNA molecule
and production of that protein or gene. Instead, they
employ either synthetic genes made by chemical synthesis or
= artificial genes made by enzymatically copying the mRNA
isolated from the donor cell to produce cDNA sequences.
One reason that natural DNA has not been pre-
viously employed directly in recombinant DNA synthesis of
proteins is that natural DNA's from most higher organisms
and at least some animal viruses contain Hintrons" or
additional nucleotide sequences as part of the gene. These
= introns do not form part of the final message of the gene.
Instead, they are removed in vivo in higher organisms by
special processing enzymes acting upon the primary trans-
cription product to afford the ultimate message (mRNA) of
the gene. Bacteria are presumed to be unable to process
CA 1341644 1979-12-21
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such introns so that natural DNA would not be expected to -
be expressed in bacterial hosts and the desired proteins
would not be expected to be produced by these hosts.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a simplified diagram re-Presenting the
structure of the endogenous DNA of a Dane particle. It
displays the two complementary DNA strands, strand a and b,
.the nick in strand a and the gap in strand b and the closing
of that gap by DNA polymerase reaction.
Figure 2 is a schematic outline of the preferred
embodiment of the process of this invention: fragments of
the endogenous DNA isolated from the Dane particle are
fused to the E. coli penicillinase gene on the plasmid
=
'pBR322 at the Pst I site. After transformation into E.
coil HB101, the recombinant DNA molecule pBR322-Pst I dG:
HBV-KpnI dC directs the synthesis of polypeptides display-
ing HBV antigenicity.
Figures 3-9 show the nucleotide sequence deter-
mined for a part of a hepatitis B virus genome in accord-
ance with this invention. The sequence is displayed with
the stop codons in the three reading frames noted above it.
It is arbitrarily numbered from the initiation codon for
the gene coding for HBcAg as determined in this invention.
Nucleotides -80 to -1 represent the leader sequence which
precedes this gene. Nucleotides 1-549 represent the nucleo-
tide sequence for the gene coding for hepatitis B virus
core antigen, the amino acid sequence (reading frame 1) of
this antigen being depicted above that sequence. Nucleo-
tides 1437-2114 represent the nucleotide sequence deter-
mined for the gene of hepatitis B virus surface antigen,
the amino acid sequence (reading frame 3) of this antigen
being depicted above that sequence. Various restriction
endonuclease recognition sites in these genes are also
depicted in Figures 3-9.
Figure 10 is a schematic outline of a process of
this invention wherein a recombinant DNA molecule which is
capable of producing a polypeptide displaying hepatitis B
CA 1341644 1979-12-21
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I.. '
11) -7-
virus core antigenicity is fragmented and a fragment
therefrom attached to an improved expression control
sequence, the lac system.
5 Figure 11 is a schematic outline of a pro-
cess of this invention wherein a recombinant DNA
molecule of this invention is fragmented and con-
nected to a fragment of phage DNA for use in lyso-
genizing host cells and thereby increasing the
number of gene copies therein.
Figure 12 is a schematic outline of another
embodiment of the process of this invention: a re-
combinant DNA molecule of this invention which does
not produce a polypeptide displaying hepatitis B
virus core antigenicity is fragmented to remove the
structural gene for HBsAg and this structural gene
employed to produce a recombinant DNA molecule which
produces HBsAg in an appropriate host.
BEST MODE OF CARRYING OUT THE INVENTION
20 In order that the invention herein described
may be more fully understood, the following detailed
description is set forth.
In the description the following terms are
employed:
25 Nucleotide--A monomeric unit of DNA or RNA
consisting of a sugar moiety (pentose), a phosphate,
and a nitrogenous heterocyclic base. The base is
= linked to the sugar moiety via the glycosidic carbon
(1' carbon of the pentose) and that combination of
30 base and sugar is a nucleoside. The base character-
izes the nucleotide. The four DNA bases are adenine
("A"), guanine ("G"), cytosine ("C") and thymine
("T"). The four RNA bases are A, G, C and uracil
("U").
35 DNA Sequence--A linear series of nucleo-
tides connected one to the other by phosphodiester
bonds between the 3' and 5' carbons of adjacent
pentoses.
Codon--A DNA sequence of three nucleotides
CA 1341644 1979-12-21
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(a triplet) which encodes through mRNA an amino acid,
a translational start signal or a translational ter-
mination signal.
=
=
CA 1341644 1979-12-21
1111
For example, example, the nucleotide triplets TTA, TTG, CTT,
CTC, CTA and CTG encode for the amino acid leucine
("Leu"), TAG, TAA and TGA are translational stop
signals and ATG is a translational start signal.
Reading Frame--The grouping of codon's
during translation of mRNA into amino acid sequences.
During translation the proper reading frame must be
maintained. For example, the sequence GCTGGTTGTAAG
may be translated in three reading frames or phases,
each of which affords a different amino acid sequence:
GCT GGT TGT AAG--Ala-Gly-Cys-Lys
G CTG GTT GTA AG--Leu-Val-Val
GC TGG TTG TAA A--Trp-Leu-(STOP)
Polypeptide--A linear series of amino acids
connected one to the other by peptide bonds between
the q-amino and carboxy groups of adjacent amino acids.
Genome--The entire DNA of a substance. It
includes inter alia the structural genes encoding
for the polypeptides of the substance, as well as
operator, promoter and ribosome binding and interac-
tion sequences including sequences such as the Shine-
Dalgarno sequences.
Structural Gene--A DNA sequence which encodes
through its template or messenger RNA ("mRNA") a
sequence of amino acids characteristic of a specific
polypeptide.
Transcription--The process of producing
mRNA from a structural gene.
Translation--The process of producing a
polypeptide from mRNA.
Expression--The process undergone by a
structural gene to produce a polypeptide. It is a
combination of transcription and translation.
S Plasmid--A non-chromosomal double-stranded
DNA sequence comprising an intact "replicon" such
that the plasmid is replicated in a host cell. When
the plasmid is placed within a unicellular organism,
the characteristics of that organism are changed or
CA 1341644 1979-12-21
=
-8a-
transformed as a result of the DNA of the plasmid.
For example, a plasmid carrying
=
/-1
CA 1341644 1979-12-21
-
-9-
the gene for tetracycline resistance (TetR) trans-
forms a cell previously sensitive to tetracycline
into one which is resistant to it. A cell trans-
formed by a plasmid is called a "transformant".
Phage or Bacteriophage--Bacterial virus
many of which include DNA sequences encapsidated in
a protein envelope or coat ("capsid"). In a uni-
cellular organism a phage replicates by a process
called transfection.
Cloning Vehicle--A plasmid, phage DNA or
other DNA sequences which are able to replicate in a
host cell, characterized by one or a small number of
endonuclease recognition sites at which such DNA
sequences may be cut in a determinable fashion with-
out attendant loss of an essential biological func-
tion of the DNA, e.g., replication, production of
coat proteins or loss of promoter or binding sites,
and which contain a marker suitable for use in the
identification of transformed cells, e.g., tetra-
cycline resistance or ampicillin resistance. A
cloning vehicle is often called a vector.
. Cloning--The process of asexual reproduc-
= tion.
Recombinant DNA Molecule--A hybrid DNA
sequence comprising at least two nucleotide sequences,
the first sequence not normally being found together
in nature with the second.
Expression Control Sequence--A DNA sequence
of nucleotides that controls and regulates expression
of structural genes when operatively linked to those
genes.
Referring now to Figure 1, we have shown
therein a simplified diagram of the endogenous DNA
= of a Dane particle. Dane particles are detectable
in the blood plasma of some patients with hepatitis B
viral infections (D. S. Dane and C. H. Cameron,
"Virus-Like Particles In Serum Of Patients With
Australia-Antiaen Assoniatpd Pann+4*4011 mu.
TWA-
CA 1341644 1979-12-21
4110
-9a-
1, pp. 695-698 (1970)). This particle is thought to
be identical or closely related to the infective
virion of hepatitis B. While its size is known
(42 nanometers in diameter), its structure is not
CA 1341644 1979-12-21
4110
-10-
fully understood. Generally, the Dane particle is
believed to consist of an outer layer and an inner
core. The outer layer of the particle generally
contains a polypeptide known as hepatitis B surface
antigen ("HBsAg"). The inner core (27 nanometers in
diameter) contains a second polypeptide, hepatitis B
core antigen ("HBcAg"), as well as an endogenous
double-stranded, circular, DNA molecule of 2.1x106
daltons, which contains a single-stranded gap of
varying length. A third polypeptide, 'e' antigen
("HBeAg"), may also be associated with the Dane par-
ticles. In addition, the Dane particle is believed .
to include an endogenous DNA dependent DNA polymerase
that partially closes the single-stranded gap in the
endogenous DNA by a polymerase reaction employing
that DNA as a primer/template. The structure and
properties of the Dane particle and more especially
its DNA have been the subject of several analyses.
E.g., W. S. Robinson, "The Genome of Hepatitis B
Virus", Ann. Rev. Microbiol., 31, pp. 357-377 (1977).
However, the actual structure of the various antigens
or their genes had not been determined.
The Dane particle's endogenous DNA has
been isolated by extraction from the Dane particle.
It consists of one nucleotide strand of constant
length having a small nick or opening in it (Fig-
ure 1, strand a, - 3000 nucleotides) and a second
nucleotide strand of somewhat variable length (Fig-
ure 1, 'strand b, - 2000 nucleotides). The second
strand is complementary with the first and overlaps
its nick to complete a circular structure by hydrogen
bonding between complementary bases in the standard
Watson-Crick manner. This overlap is apparent from
Figure 1. The DNA polymerase of the Dane particle
appears to use the second strand of the endogenous
DNA as a primer and the first strand as a template
to fill in the variable gap with nucleotides which
,i
CA 1341644 1979-12-21
rc =
4110
-10a-
are complementary to the nucleotides of the first
strand and afford a double-stranded DNA of about
3000 nucleotide pairs.
=
CA 1341644 1979-12-21
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4111. A METHOD OF PREPARATION OF HEPATITIS B VIRUS DNA
Human serum from a,single H,BsAg positive, HBeAg
positive donor (serotype adyw) was diluted with an equal
volume of buffer (0.1 M tris-HC1, 0.1 M saline (NaC1), 0.1%
(weight/volume as used throughout 2-mercaptoethanol, 0.1%
bovine serum albumin, 0.001 M EDTA) at pH 7.4. This was
centrifuged at 35,000 r.p.m. for 2 hours at 4 C. The
pellet thus obtained was resuspended in 200 ul of the same
buffer and layered at the top of a centrifuge tube contain-
ing 20% sucrose. The suspension was centrifuged at 40,000
r.p.m. for 2 hours at 4 C. The pellet thus obtained was
again resuspended in 100 ul of buffer (0.1 M tris-HC1,
0.1 M saline) at pH 7.5.
= The resultant DNA containing-pellet was then
labelled with 3H or 32P as described by P. M. Kaplan et
al., "DNA Polymerase Associated With Human Hepatitis B
Antigen", J. Virol., 12, pp. 995-1005 (1973) to facilitate
= tracing it through subsequent steps of the process. This
labelling results from the reaction of the concentrated
Dane particles and 3H-or 32P-labelled deoxynucleoside
triphosphates (dNTIlis) for 4 hours at 37 C. After this DNA
polymerase reaction, which results in partially closing the
single-stranded gap in the DNA (See Figure 1), the labelled
DNA material was layered at the top of a centrifuge tube
containing 30% sucrose and centrifuged at 42,000 r.p.m. for
3 1/2 hours at 4 C.
The DNA was then extracted with phenol from the
resulting pellet using the procedure of L. I. Lutwick and
W. S. Robinson, ItroNA Synthesized In The Hepatitis B Dane
Particle DNA Polymerase Reaction", J. Virol., 21, pp. 96-
104 (1977). The extracted DNA was then dialyzed against a
solution of 0.01 M tris-HC1, 0.001 M EDTA (pH 8.0) to
eliminate the phenol solvent. The isolated DNA was then '
ready for restriction enzyme digestions. It displayed a ,
specific radio activity of Ad 108cpm4pg. The above
discussed process is schematically depicted in Figure 2.
sf
CA 1341644 1979-12-21
1116
= -12-
CLONING OF HEPATITIS B VIRUS DNA
A wide variety of host-cloning vehicle combina-
tions may be usefully employed in cloning the double-
stranded DNA isolated as above. For example, useful clon-
ing vehicles may include chromosomal, non-chromosomal and
synthetic DNA sequences such as various known bacterial
plasmids such as pBR322, other E. coli plasmids and their
derivatives and wider host range plasmids such as RP4,
phage DNA such as the numerous derivatives of phage A,
e.g., NM899 and vectors derived from combinations of plas-
mids and phage DNA's such as plasmids which have been
modified to employ phage DNA expression control sequences.
Useful hosts may include bacterial hosts such as E. coli
.X1776, E. coli, X2282, E. coli HB101 and E. coli MRC1 and
strains of Pseudomonas, Bacillus subtilis and other bacil-
li, yeasts and other fungi, animal or plant hosts such as
animal or plant cells in culture and other hosts. Of
_
course, not-all hosts may be equally efficient. The par-
ticular selection of host-cloning vehicle combination may
be made by those of skill in the art after due considera-
tion of the principles set forth above without departing
from the scope of this invention.
Furthermore, within each specific vector, various
sites may be selected for insertion of the isolated double-
stranded DNA. These sites are usually designated by the
restriction enzyme or endonuclease that cuts them. For
example, in pBR322 the Pst I site is located in the gene
for penicillinase between the nucleotide triplets that code
for amino acids 181 and 182 of the penicillinase protein.
This site was employed by Villa-Komaroff et al., supra, in
their synthesis of protein displaying rat proinsulin anti-
genic determinants. A Hind II endonuclease recognition
site is between the triplets coding for amino acids 101 and
102 and a Tact site at the triplet coding for amino acid 45
of that protein in pBR322. In similar fashion, the Eco RI
endonuclease recognition site for this plasmid is located
between the genes coding for resistance to tetracycline and
CA 1341644 1979-12-21
111,
-13-
ampicillin, respectively. This site was employed by
Itakura et al. and Goeddel et al in their recombinant DNA
synthetic schemes, supra. Figures 2 and 11 display for
illustrative purposes some of the restriction sites in
plasmid pBR322 and phage )1/4 NM989.
The particular site chosen for insertion of the
selected DNA fragment into the cloning vehicle to form a
recombinant DNA molecule is determined by a variety of ,
factors. These include size and structure of the polypep-
tide to be expressed, susceptibility of the desired poly-
peptide to endoenzymatic degradation by the host cell
components and contamination by its proteins, expression
characteristics such as the location of start and stop
.codons, and other factors recognized by those of skill in
the art. Since the expression process is not fully under-
stood, none of these factors alone absolutely controls the
choice of insertion site for a particular polypeptide.
Rather the site chosen effects a balance of these factors
and not all sites may be equally effective for a given
protein.
Although several methods are known in the art for
inserting foreign DNA into a cloning vehicle to form a
recombinant DNA molecule, the method preferred in accord-
ance with this invention is displayed in Figure 2. It is
characterized by cleaving the Dane particle DNA with a
restriction endonuclease, attaching poly-deoxyC sequences
to the 3' termini (named by convention from the deoxyribose
carbon of the DNA sugar backbone) of the cleaved DNA and
linking the elongated DNA to a cloning vehicle which has
been cut at a particular site by a restriction endonuclease
and elongated at the 3' termini of that cut with poly
deoxyG sequences, that are complementary to the poly-dC
sequences of the cleaved DNA. The complementary character
of the 3' tails of the DNA and cloning vehicle permit
cohesion of those termini.
.To be useful in the process of this invention a
restriction enzyme chosen to cleave the HBV DNA should not
CA 1341644 1979-12-21
411,
-14-
cleave the HBV DNA within an essential part of the
gene that codes for polypeptides displaying HBV anti-
genicity, and most suitably it should cleave the DNA
at a limited number of sites. Restriction enzymes
that have been employed in this invention include
<ipri I, Bgl II, Barn HI, Ava I and Eco RI. Other re-
striction endonucleases may be similarly useful in
accordance with this invention. Their selection may
be made by those of skill in the art on due consider-
ation of the factors set out above without departing
from the scope of this invention. Figures 3-9 dis-
play some of the restriction endonuclease recognition
sites in part of the HBV genome.
Of course, other known methods of inserting
DNA sequences into cloning vehicles to form recombi-
nant DNA molecules are equally useful in this inven-
tion. These include, for example, direct ligation
wherein the same restriction endonuclease is employed
to cleave the HBV DNA and the cloning vehicle. This
procedure inherently provides complementary ends for
cohesion.
It should, of course, be understood that
the nucleotide sequence or gene fragment inserted at
the selected restriction site of the cloning vehicle
may include nucleotides which are not part of the
actual structural gene for the desired protein or
may include only a fragment of that structural gene.
= It is only required that whatever DNA sequence is
inserted, the transformed host will produce a poly-
peptide displaying HBV antigenicity.
The recombinant DNA molecule containing
the hybrid gene may be employed to transform a host
so as to permit that host (transformant) to express
= the structural gene or fragment thereof and to pro-
duce the polypeptide or portion thereof for which
the hybrid DNA codes. The recombinant DNA molecule
may also be employed to transform a host so as to
CA 1341644 1979-12-21
L
....,,,
III,
-14a-
permit that host on replication to produce additional
recombinant DNA molecules as a source of HBV struc-
tural genes and fragments thereof. The selection of
_
-
=
CA 1341644 1979-12-21
-15-
an appropriate host for either of these uses is controlled
by a number of factors recognized by the art. These in-
clude, for example, compatibility with the chosen vector,
= toxicity of the co-products, ease of recovery of the
desired polypeptide, expression characteristibs, biosafety
and costs. Again, since the mechanisms of expression are
not fully understood, no absolute choice of host may be
made for a particular recombinant DNA molecule or polypep-
tide from any of these factors alone. Instead, a balance
of these factors must be struck with the realization that
not all hosts may be equally effective for expression of a
particular recombinant DNA molecule.
In the present synthesis, the preferred cloning
.vehicle is the bacterial plasmid pBR322 and the preferred
restriction endonuclease site therein is the Pst I site
(Figure 2). This plasmid is a small (molecular weight
approx. 2.6 megadaltons) plasmid carrying resistance genes
' to the antibiotics ampicillin (Amp) and tetracycline (Tet).
The plasmid has been fully characterized (F. Bolivar et
al., "Construction and Characterization of New Cloning
Vehicles II. A Multipurpose Cloning System", Gene, pp. 95-
113 (1977)). 'The preferred host in accordance with this
invention is E. coli HB101.
1. Preparation of dc-elongated Dane particle DNA
In actual practice of the preferred embodiment of
this invention, the DNA isolated as above from the Dane
particles was digested with restriction endonuclease Kpn I
(approximately 20 ng DNA in 10)11 10 mM tris-HC1, pH 7.5,
10 mM MgCl2, 10 mM 2-mercaptoethanol, 40 mM NaCl, 0.5)11
enzyme preparation) at 37 C for 90 minutes and the re-
striction enzyme inactivated by heating at 70 C for 5
minutes. Poly-deoxyC sequences (for illustration purposes
only depicted as CCC in Figure 2) were attached to the 3'
termini of the digestion products by standard reaction with
= 35 polynucleotide terminal transferase after removing the
residual protein by extraction with phenol and chloroform
(204p1 each). Phenol was removed from the aqueous phase by
CA 1341644 1979-12-21
-16-
extraction with ether, and the DNA was precipitated by
addition of 31 sodium acetate, pH 6.5 (5).11) and cold
ethanol 0.1 ml. After storage at -70 C for one hour, the
precipitated DNA was recovered by centrifugation at 10,000
r.p.m. for 20 minutes and the pellet dissolved in 10 mM
tris-HC1 pH 7.5 (10)p1). To this was added 3)11 of 400 mM
potassium cacodylate, pH 7.0, 4 mM 'cobalt chloride, 4 mM
deoxycnosine triphosphate (dCTP), 200 ,pg/m1 bovine serum
albumin and the mixture incubated at 27 C for 10 minutes
with 0.5,p1 of polynucleotide terminal transferase
(6000 u/ml) and the reaction stopped by addition of 50 mM
EDTA (1 pl). '
2. Preparation of Pst I Cleaved,
dG-elongated pBR322
The plasmid pBR322 was digested with restriction
endonuclease Pst I (for which the plasmid contains one
target) and the products purified by phenol extraction and
ethanol precipitation in the manner described above for HBV
DNA. Poly-dG sequences (for illustration purposes only ,
depicted as GGG in Figure 2) were added to the 3' termini
of linear pBR322 molecules by terminal transferase as
described for the addition of poly-dC sequences to HBV
except that the reaction was carried out at 37 C.
3. Cohesion of G-elongated pBR322
and C-elongated HBV DNA
Equimolar amounts of the pBR322-poly-dG and HBV
DNA-poly-dC were then mixed together and the complementary
sequences allowed to link by incubation in 100 mM NaC1, 50
mM tris-HC1, pH 7.5, 5 mM EDTA (TNE), (50,p1) at 65 C for
one hour, followed by 47 C for one hour, 37 C for one hour
and 20 C for one hour and an equal volume of TNE and 20 ,11
of 100 mM MgC12, 100 mM CaCl2, 100 mM tris-HC1, pH 7.5 was
added.
4. Transformation of E. coli HB 101
Cultures of E. coli HB 101 competent, for trans-
formation were prepared as described by E. M. Lederberg and
CA 1341644 1979-12-21
;
-17-
S. H. Cohen, "Transformation Of Salmonella typhimurium
By'Plasmid Deoxyribonucleic Acid", J. Bacteriol., 119,
pp. 1072-1074 (1974). 0.1 ml portions of the cells
were mixed with 25 pl of the annealed DNA preparation
5 and incubated at 0 C for 20 minutes and then at 20 C
for 10 minutes before plating on L-agar plates con-
taining tetracycline (50 pg/m1) for overnight incu-
bation at 37 C. Since plasmid pBR322 includes the
gene for tetracycline resistance, E.coli colonies
10 which have been transformed with that plasmid will
grow in cultures containing that antibiotic to the
exclusion of those E.coli colonies not so transformed.
Therefore, growth in tetracycline containing culture
permits selection of properly transformed hosts.
15 5. Screening E.coli Colonies
by Hybridization
Bacterial colonies grown on the incubated
tetracycline containing L-agar plates were tested
for sensitivity to ampicillin. The plasmid pBR322
20 includes the gene for ampicillin resistance. This
gene is the proposed site of hybrid gene insertion.
Therefore, colonies which have been transformed with
a plasmid having DNA inserted at the chosen recogni-
tion site will be sensitive to ampicillin, but will
25 retain their resistance to tetracycline. E.coli
colonies that were sensitive to ampicillin but resist-
ant to tetracycline were picked onto a disc of Milli-
pore (Reg. Trade Mark) cellulose nitrate filter sup-
ported on L-agar plates containing tetracycline.
30 After overnight growth at 37 C, the Millipore (Reg.
Trade Mark) filter was transferred to ,fresh L-agar
plates containing tetracycline and chloramphenicol
(150 pg/m1) and incubated for a further several hours
at 37 C in order to amplify the number of copies of
35 the plasmid within the cells (Figure 2). The Milli-
pore (Reg. Trade Mark) filters were then used for
colony hybridization as described by M. Grunstein
and D. S. Hogness, "Colony Hybridization: A Method
For The Isolation Of Cloned DNAs That Contain A Speci-
40 fic Gene", Proc. Natl. Acad. Sci. U.S.A., 72,
pp. 3961-3965 (1975) with 32P-labelled HBV DNA pre-
pared previously as the probe.
r-
CA 1341644 1979-12-21
,1
4110 -18-
Radioautography of the filters revealed the presence
of colonies that contain DNA sequences complementary
to authentic HBV DNA.
6. Detection of Colonies Synthesizing
5 Polypeptides with HBV Antigenicity
Colonies that were resistant to tetracycline,
sensitive to ampicillin and hybridized with authentic
HBV DNA were tested for their ability to produce at
least one polypeptide displaying HBV antigen speci-
10 ficity or antigenicity. Colonies were again picked
onto Millipore (Reg. Trade Mark) filters supported on
L-agar plates containing tetracycline and incubated
at 37 C overnight. A second plate of L-agar was
covered with a culture of E.coli C600 (0.1 ml in
= 15 2 ml soft agar) infected with a virulent strain of
bacteriophage X vir (multiplicity of infection of
about 1) and also incubated at 37 C overnight by
which time the bacterial lawn was confluently lysed
(i.e. substantially all of the hosts' cell walls had
20 burst) by the phage (X vir). =The Millipore (Reg.
Trade Mark) filter containing cells transformed in
accordance with this invention was lifted from its
plate and placed, colonies downwards, in contact
with the surface of the L-agar plate of E.coli C600
25 that had been lysed with X vir. This contact was
continued for about 10 minutes to permit infection
of the cells residing on the Millipore (Reg. Trade
Mark) filter by X vir. The Millipore (Reg. Trade Mark)
filter was then transferred to a fresh plate of L-agar
30 and incubated at 37 C for a further 5 hours. Meanwhile
polyvinyl discs were coated with HBV antibodies as
described by S. Broome and W. Gilbert, "Immunological
Screening Method To Detect Specific Translation Pro-
ducts", Proc. Natl. Acad. Sci. U.S.A., 75, pp. 2746-
35 2749 (1978). The Millipore (Reg. Trade Mark) filters
on which the transformed bacterial colonies were now
obviously lysed were placed with the colonies in
contact with the coated polyvinyl discs and kept at
4 C for 3 hours. This contact results in any HBV
40 antigens lysed from the cells on the Millipore (Reg.
Trade Mark) filter binding with the HBV antibodies
of the disc. The coated discs were separated from
the Millipore (Reg. Trade Mark)
CA 1341644 1979-12-21
1
4110 -19-
filter and then washed and incubated with 125I-labelled
HBV antibodies. This incubation results in radio-
active labelling of those sites on the disc where
HBV antigens from the Millipore (Reg. Trade Mark)
5 filter had previously become bound by the HBV anti-
bodies of the disc. After washing and radioauto-
graphy, as described by Broome and Gilbert, supra,
colonies that had produced polypeptides with HBV
antigen specificity were located by reference to the
10 radioautograph.
USE OF POLYPEPTIDES AND GENES PRODUCED
FROM RECOMBINANT DNA MOLECULES IN DETECTING
THE PRESENCE OF HBV ANTIBODIES IN HUMANS
Polypeptides displaying HBV antigenicity
15 and the structural genes and fragments which code
therefor may be used in methods and kits designed to
detect the presence of HBV antibodies in humans and
therefore recognize humans and blood samples which
have been infected by this virus.
20 For example, the HBcAg produced by hosts
transformed by recombinant DNA molecules of this
invention can be used in the immunological diagnostic
tests currently available for hepatitis B virus
detection, that is radioimmunoassay or ELISA (enzyme
25 linked immunosorbent assay). In one type of radio-
immunoassay anti-core antigen antibody, raised in a
laboratory animal, is attached to a solid phase, for
example, the inside of a test tube. HBcAg is then
added to the tube so as to bind with the antibody.
30 To the tube coated with the antigen-antibody complex
is added a sample of the patient's serum, together
with a known amount of HBV anti-core antibody labelled
with a radioactive isotope such as radioactive iodine.
Any HBV antibody in the patient's serum will compete
35 with the labelled antibody for the free binding sites
on antigen-antibody complex. Once the serum has
been allowed to interact, the excess liquid is removed,
the test tube washed, and the amount of radioactivity
measured. A positive result, i.e. that the patient's
40 serum contains HBV antibody, is indicated by a low
radioactive count. In one type of Elisa
CA 1341644 1979-12-21
-20-
test, a microtitre plate is coated with HBcAg and to this
is added a sample of patient's serum. After a period of
incubation permitting interaction of any antibody with the
antigen, the plate is washed and a preparation of anti-human
antibodies, raised in a laboratory animal, and which are
linked to an enzyme label is added, incubated to allow
reaction to take place, and the plate is then rewashed.
Thereafter, enzyme substrate is added to the microtitre
plate and incubated for a period of time to allow the
enzyme to work on the substrate, and the adsorbance of the
final preparation is measured. A large change in adsorbance
indicates a positive result.
IN VIVO ACTIVITY OF POLYPEPTIDES
PRODUCED FROM RECOMBINANT DNA MOLECULES
To test the biological activity of the antigenic
polypeptides translated from the recombinant DNA molecule
in the E. coli of this invention, sterile extracts of the
bacterial cells which were shown to express HBcAg were
injected into rabbits after mixing with an equal volume of
Freund's adjuvant. Two animals received the crude bacte-
rial extract and two were given samples of the same extract
after fractionation on a Sephadex 650 column. Additional
injections of the same sample, which had been frozen and
stored (retaining full antigenic activity) were given two
and five weeks after the first injection. The animals were
bled at intervals several weeks after initial injection.
Immunodiffusion experiments, using the procedure
described by 0. Ouchterlony, "Immunodiffusion and Immuno-
electrophoresis", in Handbook of Experimental Immunology
(D. W. Weir ed.) (Blackwell Scientific Publications, Oxford
and Edinburgh) chap. 19 (1967), were performed to compare
the rabbit sera (antibodies) to human hepatitis B virus
core antibody ("HBcAb") using HBcAg derived from human
liver (B. J. Cohen and Y. E. Cossart, "Application Of A
Screening Test For Antibody To Hepatitis B Core Antigen",
J. Clin. Path., 30, pp. 709-713 (1977)). All four rabbit
CA 1341644 1979-12-21
. .
Ill
' -21-
.,_
sera gave precipitin lines with HBcAg identical to those
formed between HBcAg and the HBcAb derived from human
sources. Therefore, the core antigen synthesized in E.
coli with the recombinant DNA molecules of this invention
is serologically active in vivo. This activity establishes
the feasibility of compositions and methods using viral
antigens synthesized in microbial cells for the stimulation
of antibody formation in humans. Such compositions and
methods are characterized by the polypeptides produced in
hosts transformed by recombinant DNA molecules prepared in
accordance with this invention. These polypeptides would
be employed alone or with well known pharmaceutically
acceptable carriers such as saline solutions or other
.additives recognized by the art for use in compositions and
methods for the treatment and prevention of viral infec-
tions in humans.
As noted previously, restriction enzymes other
than the Kpn I/Pst I combination described above may be
usefully employed in preparing recombinant DNA molecules of
this invention. Other restriction sites in plasmid pBR322
and part of the HBV genome are depicted in Figures 2 and 3,
respectively. In illustration of these alternative, but
less preferred embodiments, the following examples are set
forth.
BAN HI, ECO RI, BGL II--PST I COMBINATIONS
The HBV DNA fragments produced by the use of
Barn HI Eco RI and Bgl .II, unlike those produced by Kpn I,
could not conveniently be directly tailed for cohesion
owing to the presence of short 5' single-stranded pro-
jections. They were therefore treated with )s exonuclease
to remove these projections before the addition of poly-dC
sequences at 3' termini. This was done by incubation of
the restricted DNA (10 )1l of a solution as described
earlier) with 15 pl 100 mM sodium glycinate, pH 9.-5, 10 mM
MgCl2, 100 pg/m1 bovine serum albumin, 5 pl ). exonuclease
at 0 C for 1.5 hours. The mixture was then extracted with
CA 1341644 1979-12-21
= 1111 -22-
phenol and chloroform and the process continued. In the
preparation in which Barn HI was used, subsequent radioim-
munoassay confirmed the production of polypeptides with HBV
antigen specificity.
DIRECT LIGATION: ECO RI--ECO RI AND BAN HI
Instead of tailing the DNA fragments for cohe-
sion, as described above, direct ligation of the gene
fragments may be employed in the processes of this inven-
tion. For example, in two further preparations HBV DNA (20
ng) was restricted with restriction endonuclease Eco RI or
Barn HI in 10 mM tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM 2-mer-
___
captoethanol, 50 mM NaC1 (10)41) at 37 C for 1.5 hours and
mixed in excess with pBR322 that had been incubated with
the same enzymes under identical conditions. A concen-
trated buffer solution (660 mM tris-HC1, pH 7.5, 100 mM
MgCl2, 10 mM EDTA, 100 mM 2-mercaptoethanol, 400 mM NaCl, 1
mM ATP) (2 )11) was added and the mixture incubated with T4
DNA ligase (0.5)11) at 10 C for 3 hours followed by 0 C for
at least 24 hours. The solution was diluted to 0.1 ml with
10 mM tris-HC1, pH 7.5 and used to transform competent
cultures of E. coli HB 101 as described earlier. In the
case of Barn HI prepared recombinant DNA molecules, the
colonies were tested, before screening for hybridization,
for sensitivity to tetracycline rather than to ampicillin,
because the target for Barn HI in pBR322 is within the gene
coding for tetracycline resistance and therefore successful
insertion at this recognition site results in loss of this
resistance instead of the ampicillin resistance as in the
case of insertion at the Pst I site.
In the case of recombinant DNA molecules prepared
via the Eco RI site, the colonies were tested, before
screening for hybridization, for resistance to both ampi-
cillin and tetracycline because the target for EcoRI in
pBR322 is between the genes coding tetracycline and ampi-
cillin resistance such that no inactivation of the genes
for ampicillin or tetracycline resistance occurs on hybrid
CA 1341644 1979-12-21
110
- 23 -
DNA insertion at this site.
Micro-organisms prepared by the =rocesses
described herein are exemplified by culturzs deposited
in the Culture Collection of the Microbiol=gical
Research Establishment at Porton Down on D-cember 15,
1978 and identified as DBR322-HBV-A to F.
Specifically, these cultures are
characterized as follows:
A: E. coil HB 101/pBR322-Pst I dG:HBV-Kpn I dc:
TetR Amps HBV* VA- [NCIB 11548]
B: E. coil HB 101/pBR322-Pst I dG:HBV-Bam HI dC:
TetR Amp' HBV- VA* [NCIB 11549]
C: E. coil HB 101/pBR322-Pst I dG:HBV-8g1 I dC:
Tee Amp HBV* [NCIB 1l550]
D: E coli HB 101/pBR322-Pst. I dG:HBV- PhI dC:
TetR Amps HBV* [NCIB 11551]
E: E_ coil MB 101/pBR322-Bam HI:HBV-Bam HI:
Tets Ai5 HBV* [NCIB 11552]
F: E oH MB 101/pBR322-Tco, RI:HBV-Eco RI:
TetR Amp' HBV* [NCIB 11553].
Portions of these identical cultures have .1so been
deposited in the Culture Collection of the Iational
Collection of Industrial Bacteria, Aberdee , Scotland
on December 20, 1979, and were assigned the accession
numbers shown above in brackets.
The above nomenclature for the cultures is a
description of that culture as follows: Ho t/cloning
vehicle - restriction site in cloning vehic e with
indication of elongation nucleotide (if an ) :
hepatitis B virus restriction site in hepatitis B virus
with indication of elongation nucleotide (if any) :
resistance (R) or sensitivity (S) to tetracicline
(Tet), and ampicillin (.Amp), positive hybri=ization to
HBv DNA in the colony hybridization test, s, ora, (HBV')
and production of polypeptide displaying H7 anti-
genicity, (VA-') Using this nomenclture culture
pBR322-HBV-A designates an E. coli HE, 101 culture
CA 1341644 1979-12-21
- 23a -
containing as a plasmid a recombinant DNA molecule
comprising pER322 cleaved at the .Pst I site and
elongated with a poly
=
/
CA 1341644 1979-12-21
111'
-24-
dG tail at the 3' termini attached to HBV DNA cleaved with
Kpn I and elongated with a poly-dC tail at the 3' termini,
the culture displaying resistance to tetracycline, sensi-
tivity to ampicillin, a positive HBV DNA hybridization test
and producing a polypeptide displaying HBV antigenicity.
Of course, it is to be understood that the hybrid
micro-organisms, recombinant DNA molecules and polypeptides
and methods applicable to them of this invention are not
limited to those described in the preferred embodiment
above. Instead, the hybrid organisms, recombinant DNA
molecules and polypeptides may be modified during produc-
tion or subsequently by known methods to good advantage.
For example, more efficient expression control sequences
may be utilized for transcription of the HBV genes or
hybrid genes, mutations to reduce the synthesis of unde-
sired gene products may be introduced, the protease levels
in the host cells may be reduced, thermo-inducible lysogens
containing the HBV genes may be integrated into the host
chromosome or other modifications and procedures may be
carried out to increase the number of gene copies in the
cell or to increase the cell's productivity in producing
the desired polypeptides.
Apart from their use to produce polypeptides
displaying HBV antigenicity, the hybrid micro-organisms of
this invention are also useful for the production of large
quantities of DNA containing all or part of the genome for
hepatitis B virus. For example, amplification, e.g., by
addition of chloramphenicol to the growth medium when the
cell density has reached a suitable level, or the use of
mutations to prevent lyais in bacteriophage hybrids that
contain HBV sequences permit the preparation of HBV DNA in
amounts previously unavailable.
HBV DNA prepared in this way may be used to
determine the nucleotide sequence of the genome and from
that the gene and amino acid sequences of the HBV antigens
and structural genes themselves. Knowledge of these
CA 1341644 1979-12-21
-25-
sequences aids in understanding the biology of the hepati-
tis B virus and permits the modifications described above
to be employed to best advantage.
DNA FRAGMENT MAPPING AND
, 5 NUCLEOTIDE SEQUENCE DETERMINATION_
1. Hepatitis B Core Antigen
A series of recombinant DNA molecules, made in
accordance with the processes described above, which
imparted to their host cells the ability to synthesize
HBcAg as detected by solid phase radioimmunoassay were
chosen for sequence analysis. Fragments were prepared from
these recombinant DNA molecules by digestion with appro-
priate restriction enzymes and the fragments labelled at
their 5' termini with [o(-32P] ATP and T4 polynucleotide
kinase. The nucleotide sequences of each fragment were
then determined by well-known chemical degradative methods
(A. M. Maxam and W. Gilbert, "A New Method For Sequencing
DNA", Proc. Nat. Acad. Sci. U.S.A., 74, pp. 560-564 (1977).
The resulting nucleotide sequence is depicted in
Figures 3-9. For reference, the-sequence is numbered from
the A of the ATG translational initiation codon of the core
gene. The nucleotide sequence of the gene for HBcAg and
the amino acid sequence for the polypeptide deduced from
this gene (Reading Frame 1) is depicted in Figures 3-9
between nucleotides 1-549. The size of the polypeptide
encoded by this gene is close to the 19000 molecular weight
observed for core antigens from Dane particles. However,
the present structural determination does not exclude the
possibility that some amino acids may be trimmed from the
amino terminus of this polypeptide in vivo during formation
of the authentic antigen. This structure also does not
take into account any other or further modifications to the
polypeptide caused by its interaction with other human
enzymes, e.g., those enzymes which glycosolate proteins.
Therefore, the polypeptide structure determined herein may
not be identical to that HBcAg found in vivo, but it will
CA 1341644 1979-12-21
= -26-
still elicit a very similar, if not identical, immuno
response.
All of the recombinant DNA molecules examined had
the HBV DNA inserted so that the core antigen gene was
maintained in the same translational phase as-the gene for
penicillin resistance of pBR322. Moreover, in the various
recombinants the HBV DNA insert began within one or two
nucleotides of the same position in the HBV sequence.
Because these recombinant DNA molecules originated from HBV
DNA digested with various restriction enzymes, e.g., Barn HI
and Kpn I, this unique attachment is surprising and may
have resulted from inadvertent breakage or polynucleotide
tailing of the HBV DNA at the nick in the endogenous DNA of
the Dane particle (Figure 1). Recombinant DNA molecules
having the HBV DNA insert in a different translational
phase did not express the HBcAg gene and no polypeptide
displaying HBV antigenicity was detected in hosts trans-
formed with such recombinant DNA molecules. Of course, it
is to be understood that such non-gene expressing hosts and
recombinant DNA molecules are still useful for producing
HBV DNA in accordance with this invention.
While it was expected that HBcAg or fragments
thereof produced through expression of the HBV DNA inserts
would be fused to the product ( fi -lactamase) of the gene
for penicillinase resistance via a small number of glycine
residues, that was not the case. Instead, the /8- lacta-
mase was in each case fused through 5-8 glycines to an
identical peptide sequence, but this sequence terminated
after 25 amino acids. In this reading frame (Frame 1,
Figure 3), a stop codon.(TAG) is followed three nucleotides
later by an initiation codon from which translation con-
tinues unhindered to give a polypeptide 183 amino acids in
length (Figures 3-8). Therefore, core antigenic activity
resides in a polypeptide of about 21,000 daltons translated
de novo from the HBV sequence within the mRNA transcribed
from the recombinant DNA molecule. This polypeptide
remains within the host cell because it is not attached to
CA 1341644 1979-12-21
-27-
the excreted penicillinase carrier protein due to the
arrangement of the stop and start codons of the HBV DNA
insert.
2. Hepatitis B Surface Antigen
The nucleotide sequence of the gene for HBsAg and
the amino acid sequence for the polypeptide deduced from
this gene (Reading Frame 3) is also depicted in Figures 6-
8, between nucleotides 1437-2114. The amino acid sequence
of this polypeptide begins at the N terminus with the
sequence met-glu-asn-ile-thr-ser. This initial amino acid
sequence was determined by D. L. Peterson et al., "Partial
Amino Acid Sequence Of Two Major Component Polypeptides Of
Hepatitis B Surface Antigen", Proc. Natl. Acad. Sci.
U.S.A., 74, pp. 1530-1534 (1977) from authentic human
HBsAg. The amino acid sequence of this protein continues
in Figures 6-8 to a stop codon 226 amino acids away. This
226 polypeptide sequence corresponds to a protein of 25,400
daltons.
The HBsAg amino acid sequence and length has been
independently confirmed by sequencing appropriate recombi-
nant DNA molecules by P. Valenzuela et al., "Nucleotide
Sequence Of The Gene Coding For The Major Protein of Hepa-
titis B Virus Surface Antigen", Nature, 280, pp. 815-819
(1979).
The nucleotide sequence determined for the recom-
binant DNA molecules of this invention shows that none of
those examined which expressed the gene for HBcAg can have
the gene for HBsAg in a position from which it could also
be expected to be expressed in an appropriate host cell.
In fact, no HBsAg was detected in the extracts of cells
transformed with those plasmids found to express the gene
for HBcAg. However, as noted, supra, knowledge of the
nucleotide sequences of these genes permits modification of
the expression process to improve its yield and effect and
to favor the expression of genes and the production of
polypeptides not previously expressed or detected.
CA 1341644 1979-12-21
lik
= ' ,
-28-
PREPARATION OF IMPROVED RECOMBINANT DNA
MOLECULES FOR PRODUCTION OF HBV ANTIGENS
The gene and amino acid sequences of these anti-
gens are useful in designing methods to increase the level
of production of antigen or gene per bacterial cell.
The level of production of a protein is governed
by two major factors: the number of copies of its gene
within the cell and the efficiency with which these. gene
copies are transcribed and translated. Efficiency of
transcription and translation (which together comprise
expression) is in turn dependent upon nucleotide sequences,
normally situated ahead of the desired coding sequence.
These nucleotide sequences or expression control sequences
, define, inter alia, the location at which RNA polymerase
interacts to initiate transcription (the promoter sequence)
and at which ribosomes bind and interact with the mRNA (the
product of transcription) to initiate translation. Not all
such expression control sequences function with equal
efficiency. It is thus of advantage to separate the speci-
fic coding sequences for the desired protein from their
adjacent nucleotide sequences and fuse them instead to
known expression control sequences so as to favor higher
levels of expression. This having been achieved, the newly
engineered DNA fragment may be inserted into a multicopy
plasmid or a bacteriophage derivative in order to increase
the number of gene copies within the cell and thereby
further improve the yield of expressed protein.
For illUstrative purposes, the sequence deter- '
mined for the gene of HBcAg has been employed in this way
to improve the production of HBcAg in E. coil. One such
process is depicted in Figure 10.
Inspection of the DNA sequence for HBcAg in
Figure 3 reveals that this gene is preceded by a target for
Alu I at nucleotide-26 (AGCT) and contains targets for
.
Eco RI* at nucleotides 22 and 1590, for Eco RII at 209
(CCTGG), for Hae III at 280 (GGCC), and for Ava II at 246
and 461 (GGACC, GGTCC). Given this complexity, it is
CA 1341644 1979-12-21
Ilb
-29-
convenient to transfer a core antigen coding sequence
to more efficient expression control sequences in
two stages. For example, digestion of a recombinant
DNA molecule (pHBV 114), prepared in accordance with
this invention, having an HBV DNA insert-of about
2350 base pairs (nucleotides), i.e., from nucleotides
-80 to about 2270 of Figure 3, with both Alu I and
Eco RI* gives, inter alia, the fragment between nu-
cleotides -26 and 22 (Fragment A) while Eco RI* diges-
tion alone gives the fragment from nucleotides 23 to
1589 (Fragment B) and low yields of a fragment from
nucleotides 23 to 576 (Fragment B'). These fragments.
may be readily identified by reference to Figures 3-4
and are depicted schematically in Figure 10. Frag-
ments A and B are fractionated by gel electrophoresis
to purify them and joined via their Eco RI* cohesive
ends to give a combined fragment (Fragment C). Frag-
ment C is now attached to a new expression control
sequence as appropriate via direct ligation to main-
tain the correct translational phase, via the 3'
tailing method described previously, or via synthetic
oligo-nucleotide linkers. This attachment not only
makes use of a better expression control sequence to
improve protein production but it also permits the
gene fragment coding for HBcAg to be attached closer
to the expression control sequence itself so as to
enhance control of that gene fragment's expression.
In like fashion, Fragments A and B' may be joined or
numerous other fragments prepared and the resultant
fragment employed as above. This process demonstrates
that only a portion of the structural gene coding
for a particular polypeptide needs to be employed
in recombinant DNA molecules of this invention.
To shorten even further the distance be-
tween the particular expression control sequence and
the initiation codon of the chosen gene fragment,
the particular fragment may be treated lightly with
CA 1341644 1979-12-21
.
_
4110
-.
-29a-
a combination of nucleases acting specifically at
or near its terminus or used in exonuclease and
polymerase-linked repair reactions to
- -
=
-
CA 1341644 1979-12-21
4111/
-30-
remove some or all of those nucleotides of the frag-
ment preceding the fragment's start codon. Alterna-
tively, a fragment, such as an Alu I fragment result-
ing from cleavage at nucleotides -26 and 30 could be
similarly shortened with exonuclease treatment or
polymerase-linked repair reactions and then cleaved
with Eco RI* to produce a fragment from between
nucleotides -26 and 1 and nucleotide 22 to permit
fusion to fragment B before attachment to the expres-
sion control sequence. A further route might involve
hybridization of fragment B, or an equivalent frag-
ment, to an appropriate single stranded template
from the parent recombinant DNA molecule for exten-
sion in a series of reactions with DNA polymerase
and a limited number of nucleoside triphosphates so
that the fragment strand could be reconstructed to
the beginning of the coding sequence. The template
strand would then be cleaved at the position of its
association to the extended fragment strand with
endonuclease Si and the fragment so obtained attached
to the expression control sequence.
Several expression control sequences may
be employed as described above. These include the
operator, promoter and ribosome binding and inter-
action sequences (including sequences such as the
Shine-Dalgarno sequences) of the lactose operon of E.
coli ("the lac system"), the corresponding sequences
of the tryptophan synthetase system of E. coli ("the
trp system"), the major operator and promoter regions
of phage X (0LPL and 0R PI), and the control region
R
of the phage fd coat protein. DNA fragments contain-
ing these sequences are excised by cleavage with
restriction enzymes from the DNA isolated from trans-
ducing phages that carry the lac or trp operons, or
from the DNA of phage A or fd. These fragments are
then manipulated in the manner outlined for the HBV
antigen sequences in order to obtain a limited popu-
CA 1341644 1979-12-21
4110
-30a-
lation of molecules such that the essential con-
trolling sequences can be joined very close to, or
in juxtaposition with the initiation codon of
CA 1341644 1979-12-21
411/
-31-
the coding sequence for the desired antigen as de-
scribed above for Fragment C.
The fusion product is then inserted as
before into a cloning vehicle for transformation of
the appropriate hosts and the level of antigen pro-
duction measured by radioimmunoassay after lysis of
the cells. Cells giving the most efficient expression
may be thus selected. Alternatively, cloning vehicles
carrying the lac, trp or X PI, control system attached
to an initiation codon may be employed and fused to
a fragment containing a sequence coding for a poly-
peptide displaying HBV antigenicity such that the
structural gene fragment is correctly translated
from the initiation codon of the cloning vehicle.
While these experiments have related to
the production of HBV core antigen specifically,
they are applicable to the improvement of the ex-
pression of other genes such as HBsAg and HBeAg genes
and fragments thereof.
Further increases in the cellular yield of
these particular antigens depend upon an increase in
the number of genes that can be utilized in the cell.
This has been achieved for illustration purposes by
insertion of recombinant DNA molecules engineered in
the way described previously into the temperate bac-
teriophage X (NM989), most simply by digestion of
the plasmid with a restriction enzyme, e.g., Eco RI
or Hind III, to give a linear molecule which was
then mixed with a restricted phage X cloning vehicle
(e.g., of the type described by N. E. Murray et al.,
"Lambdoid Phages That Simplify The Recovery Of In
Vitro Recombinants", Molec. gen. Genet., 150,
pp. 53-61 (1977) and N. E. Murray et al., "Molecular
Cloning Of The DNA Ligase Gene From Bacteriophage
T4", J. Mol. Biol., 132, pp. 493-505 (1979)) and the
recombinant DNA molecule produced by incubation with
DNA ligase. Such a procedure is depicted in Figure 11.
CA 1341644 1979-12-21
. I
µ=
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-31a-
The desired recombinant phage was then.selected by
radioimmunoassay for the particular antigen or by
hybridization with radioactively labelled HBV DNA
sequences and used to.lysogenise a host strain of
E.coli.
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CA 1341644 1979-12-21
II)-32-
Particularly useful A cloning vehicles contain a
temperature-sensitive mutation in the repression gene cI
and suppressible mutations in gene S. the product of which
is necessary for lysis of the host cell, and gene E, the
product which is the major capsid protein of-the virus.
With this system the lysogenic cells are grown at 32 C and
then heated to 45 C to induce excision of the prophage.
Prolonged growth at 37 C leads to high levels of production
of the antigen, which is retained within the cells, since
these are not lysed by phage gene products in the normal
way, and since the phage gene insert is not encapsidated it
remains available for transcription. Artificial lysis of
the cells then releases the antigen in high yield
.(Figure 11).
In addition to the E. coli systems with which
these examples have been principally concerned, the same
type of manipulations can be performed to increase the
level of antigen production in other microbial cells, such
as B. subtilis, thermophilic bacteria or yeasts and fungi,
or in animal or plant cells in culture. In the case of
B. subtilis a plasmid that carries the determinants for
penicillinase'isolated from B. licheniformis offers a
useful expression control sequence for these purposes.
PREPARATION OF IMPROVED RECOMBINANT DNA MOLECULES
FOR EXPRESSION OF THE GENE CODING FOR HBsAg
The gene and amino acid sequences determined for
HBsAg are useful in designing methods to permit expression
of the gene for HBsAg by the process of this invention.
Such expression was not previously observed in hosts trans-
formed with recombinant DNA molecules that produced poly-
peptides displaying HBV antigenicity.
The nucleotide sequence of Figures 6-8 displays
the gene coding for HBsAg between nucleotides 1437 and
2114. This gene is in a different translational phase
(Reading Frame 3) than the gene coding for HBcAg (Reading
Frame 1) in the HBV genome of Figures 3-9. Therefore, a
recombinant DNA molecule that did not produce HBcAg when
CA 1341644 1979-12-21
-33-
used to transform an appropriate host, but contained an HBV
DNA insert of about 2350 nucleotides (corresponding to
about nucleotides -80 to 2270 of the sequence of Figures 3-
8) was chosen for use in HBsAg production.
The selected recombinant DNA molecule contained,
inter alia, the entire gene coding for HBsAg. Examination
of the 5ucleotide sequence of the HBV DNA ,insert of this
recombinant DNA molecule reveals several restriction endo-
nuclease targets that permit excision of fragments contain-
ing the gene coding for HBsAg. E.g., nucleotide 1409
(Xho), nucleotide 1410 (Taq), nucleotide 1409 (Ava I) and
nucleotide 1428 (Hha I) (Figure 6). Of these the latter
(Hha I) is particularly useful because Cleavage of the HBV
DNA insert occurs between nucleotides 1430 and 1431, a cut
only six nucleotides in advance of the translational ini-
tiation codon (ATG) of the gene for HBsAg itself. In all
four cases, the excised fragment will bextend beyond the
chosen HBV DNA insert to a target for the particular
restriction endonuclease located within the nucleotide
sequence of the pBR322 portion of the recombinant DNA
molecule. Therefore, the use of these restriction endonu-
,
cleases and others similarly useful, alone or in combina-
tion, permit the excision of the gene coding for HBsAg near
but in advance of its initiation codon and after its trans-
lational termination codon. Of course, it is to be under-
stood that as illustrated in the case of the gene for
HBcAg, only fragments of the entire gene need be actually
employed in recombinant DNA molecules to produce polypep-
tides displaying HBsAg antigenicity in appropriate hosts.
ln
CA 1341644 1979-12-21
11110
those described described for the gene and gene fragments coding for
core antigen. For example, the gene fragment may be
inserted into the gene coding for penicillin resistance of
pBR322 (e.g., Pst I recognition site, Figure 2) producing
the polypeptide displaying HBsAg antigenicity-in fusion
with /7 -lactamase (the product of the penicillinase
gene), the gene fragment may be inserted between the genes
coding for penicillin resistance and tetracycline resist-
ance in pBR322 (Eco RI or Hind III recognition sites,
Figure 2) and joined there or before insertion to the lac
system expression control sequence thereby producing the
polypeptide displaying HBsAg antigenicity fused to the
/? -galactosidase protein of the lac system, the gene
fragment may be inserted into a cloning vehicle as close as
possible to the chosen expression control sequence itself
as was described previously for the gene coding for HBcAg,
or the gene fragment may be otherwise cloned as described
in accordance with this invention.
For illustration purposes, one such scheme is
depicted in Figure 12. There, the selected recombinant DNA
molecule having an HBV DNA insert extending between about
nucleotides -80 and 2270 (Figures 3-8) was fragmented by
digestion with Hha I. The resulting fragment was inserted ,
via the 3' tailing method described previously at the Pet I
site of pBR322. Hosts transformed with this recombinant
DNA molecule produced polypeptides displaying HBsAg anti-
genicity.
Other fragments (e.g., Ava I, Tag, Xho, or Pet I
(partial digest)) have also been inserted at the Pet I
recognition site or other sites in pBR322 to produce recom-
binant DNA molecules which are useful for the production of
polypeptides displaying HBsAg antigenicity or gene frag-
ments coding for HBsAg.
In addition, such gene fragments have been
inserted in pBR322 which had been cleaved by Pet I and the
resulting .fragment of the gene coding for penicillinase
CA 1341644 1979-12-21
-35-
, then chewed away from the codon coding for amino acid 182
of penicillinase or /7 -lactamase in one case to the codon
coding for amino acid 32 and in another case to the codon
coding for amino acid 13 of that polypeptide. These deny-
ative plasmids of pBR322-Pst I have also been- employed with
HBsAg gene fragments that extended from nucleotides 1447 to
1796 (Figures 6-7) to produce polypeptides displaying HBsAg
antigen;city.
The nucleotide sequence between nucleotides 948
and 1437, immediately preceding the structural gene' coding
for HBsAg, may also be included in the fragments employed
to produce useful recombinant DNA molecules for production
of polypeptides displaying HBsAg antigenicity and gene
.fragments of the structural gene for HBsAg. This nucleo-
tide sequence is referred to as the precursor sequence of
the gene. Gene fragments including both this precursor
sequence and the structural gene for HBsAg may be excised
with Alu I (nucleotide 939), Hha I (nucleotide 900) or
Hae II (nucleotide 899). In the case of Alu I and Hha I
partial digestion and separation of the resulting frag-
ments, e.g., by gel electrophoresis), is necessary because
recognition sites for these enzymes also occur within the
precursor sequence at nucleotide 1091 for Alu I and at
nucleotide 1428 for Hha II.
While we have hereinbefore presented a number of
embodiments of this invention, it is apparent that our
basic construction can be altered to provide other embodi-
ments which utilize the process of this invention. There-
fore, it will be appreciated that the scope of this inven-
tion is to be defined by the claims appended hereto rather
than the specific embodiments which have been presented
hereinbefore by way of example.
CA 1341644 1979-12-21
410 - 36 -
Micro-organisms prepared by the proce ses
described herein are exemplified by cultur is deposited
J
1
in the Culture Collection of the National Collection of
Industrial Bacteria, Aberdeen, Scotland on December 19,
1979, and identified as pBR322-HBV-G-L and
characterized as follows:
G: J ___________________ coli HB101/pBR322-Pst I dG:haff-Kon I dC
(hereinafter "pHBV114"):TetR Amps HBV" [NCIB 11554]
H: JE coli HB101/pBR322-Pst I' dG:pHBV114-Plst
I : Teta Amps HBV" VA" [NCIB 11555 and 11556]
I: E. coli HB101/((pBR322-Eco RI, Bam HI: lac promoter
sequence)-Hind III linkers:pHBV114-Hha, I Bam HI):
Tets Ampa HBV VA' [NCII3 11557]
coli HB101/pUR2*-Eco RI:pHBV114-Hha I! Eco RI
linkers:Tets Ampa HBV' VA'
NCIB 11558]
K: E. coli HB101/pBR322-Pst I dG:pHBV114-Avbõ.I dC =
TetR Amps HBV-* VA*
.(NCIB 11559]
;
L: E. coli HB101/pBR322-Pst I dG:DHBV114-Tab dC
Teti' Amps HBV" VA'
tNCIE 11560]
i
* A derivative of pBR322 having a DNA sequence
including the gene coding for the polypeptide for
tetracycline resistance replaced with a smaller DNA
sequence including the Ei_r_ coli lac system. :
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CA 1341644 1979-12-21
