Note: Descriptions are shown in the official language in which they were submitted.
BEHRINGWERKE AKTIENGBSELLSOE~T 91/B 002 - Ma 862
~2 0 ~ 3. ~ 7 ~
A Plasmodium falciParum blood-st~ge antigen, the prepara-
tion and use thereof
The invention relates to a DNA set~ence which is highly
homologous with that of a glycophorin-binding proiein,
which is called GBP 130, and has therefore been desig-
nated GBP 130 h. The invention additionally relates tt~
thP protein ~BP 130 h from Plasmodium falciparum itself
and to a process for the preparation ther~of by recom-
binant DNA techniques. Finally, the invention relate~i ~othe use of the protein GBP 130 h from Plasmodium fal-
ciparum for the preparation of malaria vaccines.
The protozoon Plasmodium falciparum - a cause of malaria
in humans - i3 a blood parasite belonging to ~he phylum
Sporozoa. Transmission from person to person is effected
exclusively by mosquitoes of the genus A_9Eh~l~L (in fact
only the females) which on biting release or take in the
parasites with the blood. For up to two weeks after the
infecting bite by a most~ito, the parasites are located
in the red blood corpuscles. In them, they assume their
ameboid forms, grow rapidly, become multin~clear and then
divide into a corresponding number of daughter indivi-
duals (called merozoites) which, liberated by disintegra-
tion of the blood corpuscle, immediately attack fresh
blood corpuscles. This reproductive process is called
~schizogony" and takes about 24 to 48 h. It is repeated,
always starting ~new, until the number of parasites
(schizonts) is so large that the body of the host xeacts
with an attack of fever to the toxic metabolic and
disintegration products of the erythrocytes and remain-
ders of schi~onts' bodies.
After a certain time~ the formation of sexual forms
(gamogony) starts, initially inside the red blood cor-
puscles, but is able to be completed only in the intes-
tine of the mosquito. Finally, after fertilization o~ thesexual forms forming in the most~uito they develop into
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what are called ~sporozoites~ which migrate from t~ 7
cavity ~o the salivary gland of the mosquito and ~here
are injected, together with the anticoa~llant saliva of
the mosquito, into the blood circulation of the person
bitten. This complete~ the generation cycle of Plasmodium
falciparum.
The incuba~ion period of tropit:al malaria, which i~
caused by Plasmodium falciparum, i5 7 to 15 days, on
averase 12 days. Tropical malaria is the most serious and
most dangerous form of malaria. In addition, it results
more often than the other forms in atypical fonms of the
disease which do not therefore immediately suggest
malaria. The prodromal signs are more pronounced and
follow one another more quickly than in tertian and
quartan malaria. The increase in temperature take~ place
suddenly, and the course of the fever is irregular. All
the systemic symptoms are considerably more severe in
tropical malaria than in the other forms. The parasitemia
rapidly increases during the course of the disease. In
extreme cases 20 to 30% of the erythrocyt~s may be
affected. Without treatment, the clinical picture rapidly
develops into a life-threatening one, with hepatomegal~,
disturbances of con ciousness, hemolytic anemia and
leukocytosis.
Since 1956, the World Health Organization of the United
Nations has organized a world-wide malaria control
campaign, which has had some great ~uccesses but also
great setbacks. The efforts of the World Health Organiza-
tion make i~ clear how considerable are the problems
associated with the occurrence of malaria for the world
population. The control of malaria has for the mos~ part
followed two lines, namely control of the vector and host
of Plasmodium falcipar~m - the Anopheles mosguito - and,
on the other hand, the development of drugs for the
treatment of malaria-infected people or people who have
to expose themselve~ to an increased risk of infection.
- 3 ~ 5
Control of the Anopheles mosquito by chemical agen~ such
as, for example, DDT has had only partial success because
the mosquitoes have in a relatively short time developed
resistance to the chemical control agen~s.
A similar resistance problem arose when various types of
drugs had been developed for the ]prophylaxis and control
of Plasmodium falciparum and other Plasmodium species.
Not all the development stages of Plasmodium which occur
in the human body respond to the same drugs. It is
therefore necessary to divide the latter into various
groups based on their mechanism action:
Action of antimalaria agents on various development
stag~s of Plasmodia
Drugs Ase~ Tissue G~x~tes Sporozoi~s
group blood forms
stages
QL~ne +~ - +
(P.vivax,
P.m~lariae)
4-km~inoline +~ - (+
Folic acid + + +
antagDnists
8-~mlox~n~line 1 ++ +
Sul~onamide + ? ~ -
9~nol~ + ? +
(meflo~ne)
~r~
The prophylactic measures likewise compri~e, on the one
hand, control of the mosquito and, on the other handr
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chemoprophylaxis. All the agents used for therapy can be
employed for chemoprophyla~is.
However, over the course of time signs of resistance by
the Plasmodia to all ~he tried ,and tested agents have
emerged. Another disadvan~age of drug therapy or prophy-
laxis compris~s the considerable side effects borne out
in the human body by the chemicals used. It is likewise
disadvanta~eous that prophylaxis must take place for as
much as æeveral weeks before and after possible contact
with Plasmodium-infected mosquitoes in order to ensure
reasonably successful protection from malaria.
This is why there has recently been world-wide discussion
of another possibility for controlling malaria. This is
the idea of vaccination against the malaria pathogen.
Considerable research effort has therefore been directed
at the identification of antigens which are suitable for
the development of a vaccine against the asexual blood
stage of Plasmodium falciparum. ~he location of antigens
on the surface of the mervzoites or of the infected
erythrocytes is regarded as one possibility. No genetic
information for parasite-encoded antigens, which might
function as carrier or receptor ("cytoadherence", "roset-
ting~), located on the surface of the infected host cell
has been found to date. In contrast to this, genes for
the antigens located on the merozoite surface tMSA I, MSA
II) have already been isolated and described in detail
(1) .
Another antigen located on the merozoite surface binds to
glycophorin (2). Glycophorin is a sialoglycoprotein on
the surface of erythrocytes. Glycophorin-binding protein
(GBP 130) which is located on the surface of the mero-
zoites i6 probably partly responsible for the recognition
of the erythrocytes by the merozoites and controls, in a
manner which is still unknown, the invasion of merozoites
into the erythrocytes (2). GBP 130 is a ~hermostable and
soluble protein which is synthesized in the trophozoite
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and schizont staqe. It is transported into the erythro-
cyte cytoplasm (3); (4); (5). GBP 130 is released into
the culture supernatant in vitro at the time the
schizonts are released. It has been shown in th.is connec-
tion that only a very small fraction of the GBP 130remains weakly associated with the merozoites ((3), (5)).
Instead of this, GBP 130 appears after release to bind to
the erythrocyte membrane, speciiically to glycophorin
(2).
Antibodies with specificity for GBP 130 are able ~o
inhibit invasion of merozoites in erythrocytes in vitro
(6). GBP 130 has likewise been described by anothex group
(5) as a 96 kDa antigen with thermoresistant properti0s.
GBP 130 is recognized by antisera which have been
obtained from saimiri monkeys immunized by drug-con-
trolled infection. These sera promote protection after
passive transfer from monkey to monkey (7); (8). Vac-
cination of the saimiri monkeys with a protein fraction
which contains GBP 130 resulted in protective Lmmunity.
The sera from the monkeys protected in this way moreover
showed a strong reaction with the 96 kDa band (9~. It was
additionally possible to show that antibodies against G~P
130 occurred exclusively in the sera from immune adults
and not in the sera from children or adults who had
already lost their immunity ~9).
The gene coding for GBP 130, which is al50 called Ag 78
or 96 tR, has been isolated from three different
Plasmodium falc parum strains ((4~, (5), (6)). It codes
for a highly conserved antigen.
The amino-acid sequence derived from the DNA sequence
comprises a chargsd N-terminal region of 225 amino acids
followed by 11 highly conserved repeats of 50 amino
acids. The gene contains a small intron which interrupts
the sequence wh.ich codes for the possible signal sequence
(10).
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The object which emerges from the abovementioned prior
art is ~o find further structures or antigens which might
be involved, in the widest sense, in the host-parasite
interaction of Plasmodium with h~uman cells. Antigens of
this type may be able to generate a protective immunity
of people against Plasmodium falcipar~m and ~hus against
malaria.
The object has been achieved by finding a DN~ with a
sequence which is called No. 1 in Seq. ID, which codes
for the protein GBP 130 h which is homologous with GBP
130.
The antigen called GBP 130 h has large homologous regions
with the already known GBP 130. Both antigens have been
investigated by the inventors with regard to the gene
structure, the gene localization and for conserved
structures in various parasite strains.
The invention embraces all DNA sequences which hybridize
with the DNA sequence shown in Seq. ID No. 1 and, at the
same time, code for the protein GBP 130 h.
The present invention likewise relates to the protein &BP
130 h and to a process for the preparation thereof by
recombinant DNA techniques.
Finally, the invention embraces the use of the protein
GBP 130 h for the preparation of a medicinal agent
agains~ Plasmodium falciparum, where this medicinal agent
is preferably a vaccine.
Identification of lambda-qtll clones which code for GB?
130 h
A genomic Plasmodium falciparum EcoRI library was
screened with a 32P-labelled, non-redundant 39mer oligo-
nucleotide which was derived from a synthetic peptide
which corresponds to the N-terminal protein ~equence of
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a possible 55 kDa surface antigen. Vaccination of aotus
monkeys with this synthetic peptide in combination with
other proteins resulted in protective ~mmunity against
Plasmodium falci~arum infection tll). The oligonucleotide
which was used for testing complies with the codon usagç
of Plasmodium falciparum according to (12). Eigh~ dif-
ferent phage clones were isolated. Sequencing of their
integrated DNA showed that none coded for the N-terminal
sequence of the 55 kDa antigen determined in (11).
However, computer analysis using the best fit prosram
from UWGCG tuniversity of wisconsin, Genetic Computer
Group) showed that one of ~he phage clones~ namely
Pfa55-1, contains a 1433 bp long DNA segment which ~hows
homology with a sequence which codes for the C-terminal
region of glycophorin-binding protein GBP 130 (6). The
protein coded by this segment (insert) wa~ therefore
called GBP 130 homologous protein, namely GBP 130 h.
Isolation of the complete_GBP 130 h gene
The inserted DNA fragment of the plasmid p55-1/RI repre-
sents parts of an intron followed by an exon with a TAA
stop codon and 261 bp of 3' non-coding region ~Seq. ID
No. 1). The inverse polymerase chain reaction method (13)
was used to isolate a 5'-overlapping subclone. Starting
from a genomic 1.25 kb Sau3AI fragment specific for GBP
130 h, a DNA sequence which extends the 5'-region of the
DNA fragment of plasmid p55-1/RI by 985 bp was amplified
(Seq. ID No. 1). The two DNA fragments represent the
complete coding region and 5' and 3' non-coding sequences
of the ~BP 130 h gene.
The sequence listed in Seq. ID No. 1 shows, besides the
nucleotide sequence of the complete GBP 130 h gene of the
Plasmodium fal iParum strain FCBR, the amino-acid
sequence of GBP 130 h derived from the DNA sequence.
Fig. 1 shows a restriction map and the structure of the
~BP 130 h gene. The coding regions (boxes) are depicted
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separated from one another by an intron sequence. The
black areas correspond to the proposed si.gnal 0equence.
The positions of the eight repeat units are indicated.
Tab. 1 shows a comparison of the amino-acid ~equence of
GBP 130 h with the amino-acid sequence of GBP 130. This
comparison was carried out using the GAP program from
UWGCG. Identity is indicated by lin s between the corres~
ponding amino acids and conserved ~mino-acid substitu
tions are indicated by colons.
Fig. 2 shows a Southern blot analysis of P. falciparum
DNA which was digested with the restriction enzymes RsaI,
HinfI, DraI and EcoRX/XbaI and was hybridized with a
32P-labelled XhoII-TaqI fragment which contains the
repetitive region of the GBP 130 h gene. The filter was
washed under mild (A) and stringent (B) conditions. In
this way GsP 130- (triangles) an~ GBP 130 h-specific DNA
fragments, and DNA fragments of a third gene (arrows)
which shows greater homology with GBP 130 h than with GBP
130, were detected.
The 5' (nucleotides 1-766) and the 3' (nucleotides
2202-2418) non-coding regions of the GBP 130 h gene are
extremely A~T rich (89% and 80.5% respectively). This has
already been described for non-coding regions of other
Plasmodium falciparum genes (14). The 155 bp-long intron
(nucleotide 9S6-1110) likewise shows a similaxly high A+T
content of 88%.
An intervening sequence of 179 bp which interrupts the
region for the probable signal seguence has been des-
cribed at the corresponding positisn for the GBP 130 gene
(10). Both introns start with GT and end with AG and axe
thus consistent with introns of other eukaryotes ~15).
The nucleotide sequences of the two introns show a
homology of 81%. This shows that the two genes are very
closely relatecl and that they therefore derive from a
common precursor gene.
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The two exons of the GBP 130 h ~ene code ~or 427 amino
acids with a calculated molecular weight of 48 260 Da.
The ATG start codon is located at position 767 and i~
flanked upstream by 4 adenine residues. This iB likewise
consisten~ with the start consensus sequences of other
Plasmodium falciparum genes (16). The N-terminus of GBP
130 h starts with a very hydrophilic region of 50 amino
acids in which lysine, serine ancl asparagine occur very
frequently. This structure has likewise been found in GBP
130 (5), (6). This region is followed by a hydrophobic
sequence of 13 amino acids which are encoded by the 3'
end of the first exon. This region, which is highly
conserved between GBP 130 and GBP 130 h, probably func-
tions as signal sequence together with the following 8iX
amino acids which are encoded by the second exon. The
predicted signal peptidase cleavage site of GBP 130 is
glycine 69 (6). An alanine residue was found at the
position in GBP 130 h corresponding to this site.
The C terminus of GBP 130 h comprises an extended repeat
region which represents 74.5% of the entire protein. This
region contains eight repeat units with 40 amino acids,
and ~his structure is very characteristic of GBP 130 h.
The repeats show only slight variations, and two of these
repeats, namely IV and V, can boast of only 39 amino
acids. The last four amino acids DE~E of repeats I, II,
VI and VII are encoded by nucleotides which are com-
plementary to the last twelve bases of the oligo-
nucleotide which was used for the screening.
As was shown by comparison of the amino-acid sequences of
GBP 130 h and ~BP 130, there is 69~ identity between the
two sequences. This corresponds to a very high degree of
homology. The main difference relates to a hi~hly charged
segment of 116 amino acids from position 110 to 225 in
GBP 130, this segment not occurring in ~BP 130 h. The
first 46 amino acids, which are encoded by the ~econd
exon, show only 54% identity between the two proteins,
which means that this segment is the most divergent
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region be~ween GBP 130 and GBP 130 h. In addition, khe
proteins differ in the number and ~he length of the
repeats. GBP 130 contains eleven repeats of 50 amino
acids, whereas ~BP 130 h shows only eight repeats with 40
amino-acid residues which correspond to amino acids 2 to
41 in the GBP 130 repea~s.
Conservation of the GBP 130 h genle
DNA fragments which corxespond to nucleotide positions
767 to 1232, which contain exon 1 and exon 2 upstream
from the repeat regions and the intervening sequence of
the GBP 130 h gene, were amplified and then sequenced.
The DNA in this case was from the Plasmodium falciparum
~;trains FCBR, FCR-3, SGE2, Il;~:2Gl, FVOR, FUl and #13. This
465 bp region of the GBP 130 h gene is identical in its
sequence for all parasite isolates which have been
analyzed to date. This shows that the GBP 130 h gene is
highly conserved.
GBP 130 h and GBP 130 are encoded by different qenes
It was possible by PCR on a genomic DNA of the Plasmodium
falciparum strain FCBR, using oligonucleotides p5 and p6
(Table), to isolate a 360 bp-long fragment which codes
for the highly charged region specific fox GBP 130.
Compared with the GBP 130 sequence of the str2in FCR-3
(6), ~his fragment shows two base-pair exchange~ which
result in substitution of amino acids: an A is replaced
by a C in position 713 of GBP 130, and an A i6 replaced
by a G in position 758. This base exchange in position
713 of GBP 130 has also been reported for the Palo Alto
isolate (10).
This GBP 130-specific probe and a 108 bp PstI-XhoII DNA
fragment from GBP 130 h (compare Fig. 1) were used for
the Southern blot analysis of Plasmodium falciparum DNA
cllt with various restriction enzymes. ~he two probes
hybridized with different DNA fragments from a Plasmodium
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falciparum strain. This shows unambiguously that the
genome of Plasmodium falciparum contains two different
genes for &BP 130 and GBP 130 h.
The _GBP_130 qene specifies a ~ene family of three dif-
ferent qenes
The repetitive region of the GBP 130 h gene was isolated
and used as probe for the Southern blot analysis of
genomic P. falciparum DNA digested with the restriction
enzymes RsaI, HinfI, DraI and ~coRI/XbaI. Three different
genes can be detected under mild washing conditions
t55~C, 2XSSC, 0.1% SDS). It was possible on the ba~is of
the known restriction maps of the GBP 130 gene (4, 5, 6)
and of the GBP 130 h gene (Fig. 1), and with the aid of
a Southern blot analysis with GBP 130 and GBP 130 h
specific DNA fragments (351 bp XbaI-SpeI fragment for GBP
130, 108 bp PstI-XhoII fragment for GBP 130 h), which was
carried out under stringent conditions, to assign unambi-
guously the GBP 130 and ~BP 130 h specific hybridization
fragments (Fig. 2A). In addition, it was pos~ible to
detect a third gene, the GBP 130 h probe cross-hybridi-
zing with an approximately 22kb EcoRI/XbaI fragment, a
1.7 kb DraI fragment, a 0.8 kb RsaI fragment and a 2.2 kb
HinfI fragment. Washing of the filter under more strin-
gent conditions (65C; 0.5XSSC, 0.1% SDS) results in no
detectable hybridization of the GBP 130 h probe with the
GBP 130 gene, but the DN~ fragments which are assigned to
the third as yet unknown gene are detected (Fig. 2B).
This shows that this gene is more homologous with the GBP
130 h gene than with the GBP 130 gene.
The GBP 130 h aene is only very weaklY xpressed in blood
staqes of_P. falciparum
Starting from poly(A)+ RNA from schi20nts, a Northern blot
analysis was carried out with a 108 bp Ps~I-XhoII frag-
ment (specific GBP 130 h gene fragment) and with a 351 bp
XbaI-SpeI fragment (specific GBP 130 gene fragment). ~he
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two fragments used for the hybridization have appxoxi-
mately the same specific radioactivity. The GBP 130 probe
detected a unique mRNA of about 6.5 kb as dominant band
after overnight exposure. Similar results have already
been described in the literature for GBP 130 (4, 5, 6).
In contrast with this, the GBP 130 h probe hybridized
with two mRNA bands of about 2.5 kb and about 2.8 kb
which were detectable only after a very long exposure
tLme of 8 days. In this case, one of the mRNAs can be
assigned to the GBP 130 h gene; the second band might
repr~sent the mRNA of the third gene of the GBP gene
family, which evidently shows high homology with the GBP
130 h gene (Fig. 2). There is a noticeable discrepancy
between the expression rate of the G~P 130 gene and that
of the gene which codes for the protein GBP 130 h: the
GBP 130 gene is transcribed with very high efficiency,
whereas the transcription rate of the GBP 130 h gene, and
evidently of the third gene of this gene family, is very
weak. Supposing that the proteins translated by these
mRNAs have approximately simil~r frequency rates, it must
be assumed that GBP 130 occurs very frequently in P.
falciparum schizonts, and GBP 130 h tends to be und~r-
represented. This different distribution of the~e
homologous proteins might be utilized for the so-called
smokescreen effect: this entails GBP 130 being released
in large quantity after the merozoites have been released
from the schizonts, and it might have the task of divert-
ing the Lmmune system, in which case the under-
represented GBP 130 h might simultaneously exert its
essential function.
Table 2 shows the ~equences of oligonucleotides which
have been constructed for the polymerase chain reaction
(PCR).
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Examples
Pre~earation of DNA and mRNA
Standard methods (17) were used for the cultivation of
the Plasmodium falc_parum strains FCBR tColombia), FCR-3
(Colombia), FVOR (Vietnam), SGE2 (Zaire), ItG2Gl (srazil)~
FUl (Uganda) and #13 (Senegal), for the enrichment of
schizonts and for the preparation of DNA and poly(A)+ RNA.
ThP analysis of DNA and mRNA of the strain FCBR by
Southern and Northern blot technology is likewise des-
cribed in the prior art (18).
Construction of a qenomic EcoRI :LibrarY
2 ~g of DNA of the Plasmodium falciparum strain FCBR wereincubated with 14 units of the restriction enz~me EcoRI
in 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 ~M dithio-
threitol and 40~ (v/v) glycerol at 37C overnight. Underthese conditions, EcoRI shows star activity. This means
that the DNA is digested at its tetranucleotides AATT.
The DNA fragments with a ~ize up to 10 kb resulting from
this were fractionated using a 0.8% agarose gel. Fra~y-
ments between 500 bp and 7 kb were electroeluted and theninserted into the vector lambda gtll by the method
described in ~19). A genomic EcoRI library of 5 x 105
recombinant phage clones was obtained in this way, and
these phages were then amplified by standard methods
(20).
Screeninq of the EcoRI library
1.5 x 105 phage clones from thi~ library were screened by
standard methods (20) using a 5'-32P-labelled oligo-
nucleotide which was derived from the N-terminal protein
sequence of a possible 55 kDa Plasmodium falciparum
surface antigen (11). The oligonucleotide had the base
sequence~ 5'-TGC TGC ATA TAC ATT TTG TGT ~TC TGC TTC TAA
TT(: ATC-3 ' .
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The oligonucleo~ide was constructed on the basis of the
codon~ most commonly used by Plasmodium falciparum (16).
Eight phage clones were i~olated and one of these, which
was called Pfa55-1, was used for the subsequent invez-
tigations. The DNA of the phage clone Pfa55-1 was diges-
ted with the restriction enzymes EcoRI and KpnI. This
resulted in a 2.4 kb fra~ment which, besides the malaria-
specific fragment, carried a 1 kb of lambda gtll region,
which was deleted by means of par~ial PvuII restriction.
The 1.4 kb fragment obtained in this way was cloned into
the pKS(+) Bluescript vector, which was digestible with
EcoRI and SmaI, resulting in the plasmid pS5-1/RI .
Isolation of a 5'-overlap~ina qene fraqment by inverse
PCR
In order to isolate the complete gene, a fragmenk of
which is contained in the phage clone Pfa55-1, the 5'
region of the gene was extended by means of inverse PCR
(13). A genomic 1.25 ~b Sau3AI fragment which extends ~he
5~ region of the inserted DNA of the phage clone Pfa55-1
by 985 bp was identified by Southern blot analysis (20~,
using a 32P-labelled 108 bp fragment (PstI/XhoII digPstion
of p55-1/RI ) as proba. 90 ~g of Sau3AI-digested P.
falciparum DNA were fractionated on a 0.8% agarose gel,
and the DNA fragments with a si~e between 1.2 and 1.3 kb
were electroeluted. The Sau3AI cleavage sites were self-
ligated and, after restriction with the enzyme PstI, the
known DNA sequences were converted to the 5' and 3' end.
50 ng of this genomic DNA and 500 ng of the oligo
nucleotides pl and p2 (Tab. 2) were u~ed for the PCR,
which was carried out under standard conditions u~ing the
Gene-AmpR kit from Perkin Elmer Cetus. The 1.25 kb frag-
ment obtained ater this was phospho~ylated at the 5'
ends. This was followed by a fill-in reaction with the
Klenow en~yme (20). This DNA fragment was then inserted
into the pKS vector which had been digested with SmaI.
The plasmid formed in this way was called pS5-1/PCR.
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DNA sequencing
Both strands of the inserted DNA fragments of the plas-
mids p55-ltRI~ and p55-1/PCR were sequenced by the dideoxy
method using the sequenase system from ~SB (Cleveland,
OH). Suitable subfragments were obtained by subcloning at
available restriction sites in~o the Bluescript vector
pRS. The sequencing data were analy~ed using the ~WGCG
program (21).
Amplification and sequencin~ of specific ~ene reqions of
various P. falciparum isolates
0.5 ~g of DNA from the P. falciparum strains FCBR, FCR-3,
SGE2, ItG2Gl, FVOR, FU1 and #13 were used in combination
with, in each case, 300 ng of the oligonucleotides p3 and
p4 (Tab. 2) to ~mplify a genomic fragment. ~he Gene-AmpR
kit from Perkin Elmer Cetus was u~ed for this. The
genomic fragments of the seven different parasite iso-
lates were phosphorylated, then sub~ected to a fill-in
reaction using the Klenow enzyme by standard methods (20)
and subsequently inserted into an SmaI cleavage site of
the vector pKS or sequencing.
Construction of a GBP 130 specific probe
Comparison of the coding sequences of GBP 130 h and GBP
130 (6) resulted in the discovery of a 351 bp-long
fragment which is present only in the GBP 130 gene. This
GBP 130 ~pecific fragment was amplified by a genomic DNA
of the P. falciparum strain FCBR, specifically using the
oligonucleotides p5 and p6 ~Tab. 2~. The 360 bp fragment
resulting ~rom this was digested with XbaI and SpeI and
then ligated into the ~ector pRS, resulting in the
plasmid pKS/GBP. The identity of the GBP 130 ~pecific
fragment was checked by DNA sequencing.
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Southern blot analysis of ~he GBP 130 and ~BP 130 h qene
The GBP 130 specific fragment was isolated from the
plasmid pKS/G~P using the xestriction en~ymes XbaI and
SpeI, and labelled with 32p by nick translation. A 108 bp-
long GBP 130 h specific DNA fra~ment was isolated analo-
gously from the plasmid pS5-l/RI~ using the restriction
enzymes Pstl and XhoII and was labelled with 32p by nick
translation. soth probes were usecl for the Southern blot
analysis by standard methods (20) of P. falciparum DNA
which was digested with the r~s1,riction enzymes DdeI,
TaqI, AluI, Sau3AI, RsaI, HinfI, DraI and EcoRI/XbaI.
Southern blot analysis of genomic P. falci~arum DNA usinq
the repetitive GBP 130 h probe
The plasmid p55-l/RI* was digested with the restriction
enzymes XhoII and TaqI, and it was possible to isolate a
965 bp DNA fragment which contains the 8 repeat units of
the GBP 130 h gene. This DNA fragment was radioactively
labelled with 3ZP by nick translation (20) and used for
~he Southern blot analysis of P. falciparum DNA which was
digested with the restriction enzymes RsaI, HinfI, Dra~
and EcoRI/XbaI. After the hybridization, which was
carried out using standard conditions (20), the membrane
was first washed in 2XSSC (2XSSC is 300 mM NaCl, 30 mM
sodium citrate), O.l~ SDS (sodlum dodecyl sulfate) at
55~C twice for 15 minutes each time and subsequently
autoradiographed for 3 h. After development of the
autoradiograph, the membrane was washed under more
stringent conditions in 0.5XSSC, 0.1% SDS at 65C twic~
for 15 minutes each time and subsequently expo~ed over-
night.
Northern blot analysis
10 ~g samples of a poly(A)+ RN~ which was isolated fromschizonts of the P. falciparum strain ~CBR were frac-
tionated using a 0.8% agarose/formaldehyde gel, then
~ .
:
- 17 ~ 75
transferred to a Gene-Screen membrane tDu Pont) using the
supplier's protocol r and hybridized with a nick-trans-
lated 108 bp-long PstI-XhoII fragment of the GBP 130 h
gene which was obtained from the plasmid p55-1/RI~, and
with a 351 bp XbaI-SpeI fragment of the GBP 130 gene
which was obtaine~ from the pla3mid pRS/GBP. The filter
was washed twice for 15 min. in 0.5XSSC, 0.1% SDS at
55C/ and autoradiographed.
Expression of a part al BP 130 h se~lence
The vector pSEM tsiotechni~ue~ 8,]pages 280-281 (22)) was
used to express a part-sequence of the plasmid p55-1/RI~,
the fusion protein carrying the 375 N-terminal amino
acids of ~-yalactosidase. The oligonucleotides p7 and p8
(Table) and 100 n~ of the plasmid DNA from p55-1/RI were
used to amplify a 680 bp fragment by PCR. The amplified
fragment was digested with SacI and PstI and then ligated
into the pSEM1 vector which had been linearized with the
same restriction enzymes. The E. coli strain DHSalpha was
transformed with the ligated plasmid. After this, colo-
nies which contained the inserted DN~ fragments of the
correct size were isolated. Single colonies were cul-
tivated overnight and then induced with 1 mM IPTG (iso
propyl thiogalactoside) for 2 h. The expression products
were analyzed by SDS polyacrylamide gel electrophore~is.
In this case i was possible for a fusion protein of
70 kDa to be expressed in high yield.
,
- 22 - 2~6~3~
Tahle 1
.
1 MRIS-~SNIESTGVS~1C:~NFNSXNCSXYSLMEVQN~NE~X~SLTSF~KN 50
11:11-1:1-111111:1111111:11111111 IlllI.li..ll.l-
1 MRLSXVSDIKSTGVSNYXMFNS~NSSXYSL-'qEVSX~NEKgNSLG~F35~X 50
51 ITLIFG~ IYVAL~GVYIC~SQYRQAADYS~RES~VLAEG~STSXRNA~TA 100
51 ILLI~GIIYWLLNAYICG3RYE~AVDYG~ESRII,AEGEDTC~RREgTT 100
.
101 LRKTRQTTL................... ...........................109
I I I I I I
lGl LRKSXQXTST~TV~TQTRXDE~NRS W TEcQKVESDSEXQXRTX~W RKQ 150
109 .'.......................... ...........................109
151 INIG~TENQKEGXNVX;;VIXKrXXXEESGXPEENXaANEASRXXEP.YASX 200
109 ............................ ...........................109
201 VSQ~?STST~SNNEVRIR~ASNQETLTS~DPEGQI~REY~DP~YRK~LE 250
~ 09 ........................... ...........................109
25i IFYXLLTNTDPNDEVERRNADNXEDLTSADP''GQIMREYAS3P~YRXcL~ 300
109 ............................ ...........................109
301 IFV~ILTNTDPNDDVcRRNADNXEDLTSADPEGQI~qREY~ADPEYRXHLE 350
710 ............................ .TS~DPEGQIMKAWAADPEYRK~LN 133
1111111111:.:1111111111:
351 V~-~Rl~TNTDP~lD~VERRNADNX_3LTSADP~G~I~REY~ADPEYRX:'L_ 400
134 VLYQILNNTDPND~L~............ .TS~DP-5QIMRAVAAD?EYRR;;LN 173
::- 11-1111111:1 11111l1111:-11111111111:
~01 lEE~ILTNTDpNDEvrRRNADN~EDLTs~Dp-GQIMREvA~Dpry~R~L~ 450
77q VLYQIL.~NTDPNDEVE........... SSADPrC-QI~q.YAY.~DPrY~X~-VNV 214
451 VF3~ILTNTD~NDEVLRRNADNX_LTSSDPEGQI:qRE~V`~ADP~Y~XuLrI 50O
.
275 LYQILN~TDPNDELr............. TSADP~rQ_~g~VAADP~'~Rr~v~v 254
: I I I I I 1'1 1 1: 1 1 1 1 1 1 1 1 1 1 1: I I I I I I I I I 1:::
501 -.XILTNTD5NDEVER~NADNXEDLTSADaEGQ_~RrY~A3P~YRKr.LE 550
25~ LYQILNr.TDSS._VE............ TSAD2_C-QIiYRAYA~D2~YRR~NV 293
:1-11-:11-- 111 II-IIlilll:-llllllllll:::
~1 Fy~ILT~TDpNDEvER~NADNxrELTssDprGQI;qREyAADpEy~Rr3r; 600
2S4 LYQIL~2TDSS.~VE............. TSADP--C-~IM~VAADP~YRXEV~ 332
601 FEKILT~TDPNDEVERRNADNgEDLT5ADPEGQIMREY~ADPEYRXr3 _I 6;0
.
333 LYQIL~NTDPNDEL_............. TSADP_r-QI;~XAYAADP~YRXF.~NV 372
:1-11-1111111:1 Illlllilll:-ll-lllllll:::
6~1 FYXILT~TDP~DEVE~RNADNR~DLTSADP~GQIMREVA53PrYRgr.LE~ 700
373 LYQIL~NTDPNDELE............. TSrDP~5QI~qKAY.~ADPrYRXn~NV 412
701 EYXILT~TDP~DDVEP~RNADNXrDLTS.;DP--C-2Il~REV~ADP~YRK~LEl /;0
. _ . .
413 LYQIL~NTDPNDESS............. 427
: I I I I I I I I I
751 FUXIL~TDP~DEVERQNADN~'EA 774
,~
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- 23 - 2 ~ ~ 1 3 7 ~
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la ~ ~ ~, H H
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p~ ~ ~ ~
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.
- ~4 -
SEQ ID NO: 1 2 0 6 1 ^ r7 ~
TYPE OF SEQUENCE: nucleotide with corresponding pro~.ein
STRANDEDNE5S: single strand
TOPOLOGY: linear
TYPE OF MOLECU~E: genome DNA
ORIGINAL 50URCE
ORGANISM: Plasmodium falciparum
IMMEDIATE EXPERIMEN~AL SOURCE
NAME OF THE STRAIN: P. falciparum FCBR
FEATURES:
from 767 to 955 BP exonl
from 1111 to 2202 ~P exon~
from 1249 to 2202 BP repetitive rlegion
PROPERTIES: gene which code~ for an antigen homologous
with the GBP130 protein
GA$CTATATT AAAAAAAATA TACAAGGAAA AGATGTG~TA AACAATATAC ATTSATATAA 60
TATATAATTA ATATAATATA ATATAATGTA GTATTTCATG AAATATATTA TGTAGGSTTG 120
ATTTAAATTT AATCATTATA TAAATATTAC ACATATGAAT AAATAAATAA ATATATATAT 180
ATATATATAA ATATATTTAC TTATATTTAG TAATTTTTAA CATTGTATAT TTAAAAAGAA 240
AATATTTTTT ATTTCATATT TTTTAATATT TTATAATAGA AAAAGATAGA TACAAAAGAA 300
CATGTGGAAA AAATATATAT TATAAAATAT ATTCTAAATT CTTTTATTAA TTTTAAGATT 360
CAATAAAAAT AATATA$C.;A TG~AATAATT AATTATTTTA ,AATCAATAG TCGTAAGTGT 420
AATACATATA TTATTTC-AG CTTCTTGTAA CTTACATAAT TATAAATATA TCATATATTC 480
TTTCAAGGAT ATGATAA.TT ATATCATTGA AAAAATATA- ATATAGTATT TATCTTTTAT 540
GAA~U~AAAC ATTGAAA.GT AATTTATGTA AAu~vuuU~A AAATTAAAAT AAAATAATAA 600
AAAAATATTT ATGTATTG.T TTTTTTTTTT ATTTTTATTT TATTATTTTA AAATATATAT 660
TTAAAAAAAA AAAAATATAT ATATATATAA TATTTATA-- TATAATTATT TTAGAAACAC 720
ACAAATTAGA AAAAACAT.iT ATATTCTTAT TTTCTTCT~; GTAAAA ATG CGT ATT 775
Met Arg Ile
TCA AAA GCA AGT AAT ATT GAA TCT ACA GGA G-T TCG AAT TGT AAA AAT 823
Ser Lys Ala Ser Asn Ile Glu Ser Thr Gly V21 Sec Asn Cys Lys Asn
; 10 15
TTC AAT TCG AAA AAT TGC TCT AAA TAT TCT T-S ATG GAA GTA CAA AAT a7
Phe Asn Ser Lys Asn Cy5 Ser Lys Tyr Ser Le~a Met Glu Val Gln Asn
ZS ;o 35
AAA AAT GAA AAG AAA CGT TCC TTA ACT TCC T-_ CAT GCC AAA AAC ATC 919
Lys Asn Glu Lys Lys Arg Ser Leu Thr Ser Phe ~is Ala Lys Asn Ile
ACA TTG ATT TTT GGA ATA ATA TAC GTA GCC T-A TTG GTATGA TAATATAAAT 971
Thr Leu Ile Phe Glv Ile Ile Tyr Val Ala Leu Leu
5; 60
AAAATATATA ATTGAATTTT TTTCTTTTTT AATTAATGAA TTTAAATATC CTTATTAGAA 1031
ATATATAGAT ACATAGATAC ATACATAAAT ATATATTTA- ATATATATAT TTTTTTCTGT 1091
TTGCATCATT TATTTTTAG GGT GTT TAT ATA TGT G~ AGC CAA TAC AAA CAA 1143
Gly Val Tyr Ile Cys Ala Ser Gln Tyr Lys Gln
- 25 - 20~ ~7~
GCT GCA GAT TAT AG~ TTT AGA G~A AGC AGA GTT T~A GCT GAA GG~ AAA 1191
Ala Ala Asp Tyr S~r Phe Arg Glu Ser Arg Val Leu Ala Glu Gly Lys
AGT ACC AGT AAA AAA AAC GCA AAA ACC GCA TTA AGA AAA ACT AAG CAA 1239
Ser Thr Ser Lys Ly~ Asn Ala Lys Thr Ala Leu Arg Lys Thr Lys Gln
100 105
ACA ACC TTA ACT AGC GCA GAT CCA GAA GGA CAA ATA ATG AAA GCC TGG 1287
Thr Thr Leu Thr Ser Ala Asp Pro Glu Gly Gln Ile Met 1ys Ala Trp
110 115 120
GCT GCT GAT CCA GAA TAT CGT AAA CAC CTA AAT GTT CTT TAC CAA ATA 1335
Ala Ala As? Pro Glu Tyr Arg Lys Hls Leu Asn Val Leu Tyr Gln Ile
125 130 135
TTA AAT AAC ACT GAT CCA AAT GAT GAA TTA GAA ACT AGC GCT GAC CCA 1383
Leu Asn Asn Thr ASD Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro
140 145 150
GAA GGA CAA ATA ATG AAA GCT TAT GCT GCT GAT CCA GAA TAT CGT AAA 1431
Glu Gly Gln }le Met Lys Ala Tyr Ala Ala ~sp Pro Glu Tyr Arg Lys
155 160 L65 170
CAC CTA AAT GTT CTT TAC CAA ATA TTA AAT AAT ACC GAC CCA AAT GAT 1479
His Leu Asn Val Le~ Tyr Gln Ile Leu Asn Asn Thr Asp Pro Asn Asp
175 180 laS
GAA GTA GAA TCT AGC GCT GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT 1527
Glu Val Glu Ser Ser Ala Asp Pro Glu Gly Gln Ile Met Lys Ala Tyr
190 195 200
GCT GCT GA. CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA 1575
Ala Ala As~ Pro Glu Tyr Arg Lys His Val Asn Val Leu Tyr Gln ~le
205 210 215
TTA AAT AAC ACC GAT CCA AAT GAT GAA TTA GAA ACT AGC GCA GAC CCA 1623
Le~ ~sn Asn ~hr Aso Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro
220 225 230
GAA GGA CAA ATA ATG AAA GCC TAC GCA GCT GAT CCA GAA TAT CGT AAA 1671
Glu Gly Gln Ile Met Lys Ala Tyr Ala Ala A5? 2ro Glu Tyr Arg Lys
235 240 2~5 250
CAT GTA AAT GTC CTT TAC CAA ATA TTA AAT CAC ACC GAC TCA AGT GAA 1719
8is Val As~ Val Leu Tyr Gln Ile Leu Asn 9is Thr Asp Ser Ser GLu
~5, 260 26;
GTA GAA AC- AGC GCA GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT GCT 176,
Val Glu Thr Ser Ala Asp Pro Glu Gly Gln Ile .~et Lys Ala Tyr Ala
270 . 275 2eo
GCT GAT CCA GAA TA. CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA TTA 1815
Ala Asp Pro Glu Tyr Arq Lys His Val Asn Val Leu Tyr Gln Ile Leu
285 ~90 295
AAT CAC ACC GAC TCA AGT GAA GTA GAA AC- AGC GCA GAC CCA GAA GGA 1563
Asn 8is Thr Asp Ser Ser Glu Val Glu Thr Ser Ala Asp Pro Glu Gly
300 305 310
CAA ATA ATG AAA GCC TAC GCA GCT GAT CCA GAA TAT CGT AAA CAC GT~ 1911
Gln Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys His Val
315 320 325 330
AAT GTC CTT TAT CAA ATA TTA AAT AAC ACT GAT CCA AAT GAT G~A TTA 1959
Asn Val Leu Tyr Gln Ile Leu Asn Asn Thr Asp ~ro Asn Asp Glu Leu
335 340 345
GAA ACC AGC GCA GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT GCA GCT 2007
Glu Thr Ser Ala Asr 2ro Glu Gly Gln Ile Met Lyr Ala Tyr Ala Ala
350 ' 355 360
, . ~ . , ~ :
: . :
.
- 26 -
GAT CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAT CAA ATA TTA AAT ~ 5
Asp Pro Glu Tyr Arg Lys Hls V~l Asn Val Leu Tyr ~ln Ile Leu Asn
365 370 375
AAC ACT GAT CCA AAT GAT GAA TTA GAA ACT AGT GCT GAC CCA GAA GGA 2103
Asn Thr ASp Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro Glu Gly
380 385 390
CAA ATA ATG AAA GCT TAT GCT GCT GAT CCA GAA TAT CGT AAA CAC GTT 2151
Gln Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys ~is V~l
395 400 405 410
AAT GTC CTT TAC CAA ATA TTA AAT AAC ACT GAT CCA AAT GAT GAA AGT 2199
Asn Val Leu Tyr Gln Ile Leu Asn Asn Thr Asp Pro Asn Asp Glu Ser
415 420 425
TCC TAAGAA TGTATCTCCC TTCG~AAAAT AAGAG-~AAAC AAAATTTGCA AATGAATTAG 2258Ser
AAAGTACGAT TATGATAATT AAGAGATGTA TGAAT'rTGAA TGTAAAAATG ACATTTTTTA 2318
TAATAACGTA CAATATTTTA ATAATTAATT ATCAA~ATG AAATATATAA TACTATTTAT 2378
GGTATTTGAT ATTATTTAGA TGAGGAAGAA AAAAGGAATT 2ql8
.
:, ;
., ' ' ; , . ~ , ,:
