Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CHIMERIC GENE FORMED OF THE DNA SEQUENCES THAT ENCODE THE
ANTIGENIC DETERMINANTS OF FOUR PROTEINS OF L. INFANTUM,
USEFUL FOR SEROLOGIC DIAGNOSIS OF CANINE LEISHMANIASIS
AND PROTEIN OBTAINED
DESCRIPTION
OBJECT OF THE INVENTION
The present specification relates to an application
for an Invention Patent, regarding a chimeric gene formed
of the DNA sequences that encode the antigenic
determinants of four proteins of L.~infantum, useful for
serologic diagnosis of canine leishmania,sis and protein
obtained. The obvious purpose of this lies in using the
protein obtained from the chimeric gene to perform an
early diagnosis of canine leishmaniasis, that can be
present in the body of a patient. This patient does not
have to be a dog but can also be a human being who
suffers from diseases that involve immuno-depression.
This achieves an accurate diagnostic that avoids current
diagnostic methods. These, in view of the fact that the
antibodies present in animal and human serum to be
analysed contain a large quantity of proteins can produce
cross reactions, and can therefore give positive results
when there is no real infection. Therefore existing
types of analysis can give rise to uncontrolled false
positive readings.
To summarise, with a view to minimising these
problems, a chimeric gene will be produced that encodes a
protein called MSPQ consisting of a chimeric product
originating from an "in vitro" synthesis of a chimeric
gene constructed "ad hoc", which contains five of the
antigenic determinants of four different proteins. The
product is configured as a highly sensitive and specific
for the diagnosis of canine Leishmaniasis.
FIELD OF THE INVENTION
This invention is of utility within the industry
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dedicated to the manufacture of pharmaceutical products
in general.
BACKGROUND OF THE INVENTION
The parasitic protozoa of the Leishmania genus are
the aetiological agents that cause Leishmaniasis, a range
of diseases that have a world-wide distribution and that
are characterised in that they give rise to a wide
variety of clinical symptoms.
The main forms of Leishmaniasis are zoonotic in
nature and humans are considered as secondary hosts.
The species denoted L. Infantum, widely distributed
throughout many Mediterranean areas is the cause of
visceral Leishmaniasis (LV) in humans and dogs.
In fact, dogs infected with L. infantum are the
main animal reserve of this parasite, particularly during
the long incubation period before the clinical symptoms
can be observed.
The epidemiological data indicate that there is a
direct correlation between the prevalence of canine
Leishmaniasis and the transmission of the parasite to
humans. For this reason, it is crucial to detect the
disease or infection early on in campaigns undertaken to
control the spread of the disease.
The parasite is transmitted to the host vertebrate
as a flagellate promastigote, by means of a bite of a fly
of the family "Phlebotominae", and the parasite enter the
cells of the mononuclear phages where they differentiate
and reproduce as amastigotes, within the phago-lisosomal
structure.
The infected cells gather in certain tissues,
mainly spleen, liver and lymph nodes. It is estimated
that around 15 million people are infected with
Leishmaniasis, and every year in the world 500,000 new
clinical cases appear in the world, mainly in the
underdeveloped and developing world.
In the south-western countries of Europe, Visceral
Leishmaniosis (VL), is a zoonotic disease caused by the
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L. Infantum species, as was mentioned earlier. Recent
data derived from epidemiological studies indicate that
there is an alarming incidence of this infection.
In Italy the reported data for incidence of VL
ranges from 14.4% to 37% according to the region.
In Portugal, more particularly in the area around
Lisbon, seropositive rates of 8.4% have been found and in
the region of the French Maritime Alps different centres
of prevalence have been found that vary between 3.2% and
17.1%.
In Spain, the prevalence of Leishmaniasis depends
on the zone being studied. In Catalonia an average
incidence rate of 9.3% has been observed although in some
hot-spots a prevalence of infected dogs of up to 18% has
been found.
On the Island of Mallorca, the incidence rate is
14%, and other rates that have been found are: 2.4% in
Murcia, 8.8% in Granada, from 10 to 15% in Salamanca,
5.25% in the province of Madrid, and 14% in Caceres.
Although the number of cases of VL in humans caused
by L. infantum can be considered relatively low, the high
percentage of patients with immuno-depression that become
infected by Leishmania could be related to the high level
of this illness in dogs.
In fact, in the South of Europe, 50% of adults that
are infected by Leishmaniasis are also patients infected
by the HIV virus. On the other hand, according to these
data of Leishmania-HIV co-infection, it has been
estimated that the level of infection (by parasites) can
be one or two orders of magnitude higher than this figure
due to the existence of a large number of undetected
infections.
A common characteristic of the different types of
Leishmania infection is that it induces a strong humoral
response in the host. Therefore, diagnostic methods based
on serological techniques are currently the most widely
used.
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It has been described that these antibodies are
detected even during the asymptomatic phase of the
disease in natural and experimental infections.
The sensitivity and specificity of these methods
depends on the type, source and purity of the antigen
used. In immunological processes that are currently
commercialised, complete promastigotes and preparations
more or less prepared from these are used as a source of
antigen. This method normally leads to cross-reactions
with serum from patients suffering from leprosy,
tuberculosis, African tripanosomiasis, Chagas disease,
malaria and other parasitosis.
The sensitivity and specificity of the serologic
methods depend on the type, source and purity of the
employed antigen. During the last years a great number of
Leishmania antigens have been characterised, some of them
can be considered as proteins specific to the parasite.
Among these proteins specific to the parasite, the
surface protease GP63, the surface glycoprotein gp46 and
the lipophosphoglicane associated KMP-11 protein deserve
a mention.
An additional group of Leishmania antigens are
formed of evolutionarily conserved proteins, such as
kinesine, thermal shock proteins, actin and tubulin.
As part of a strategy to develop a specific
serological diagnostic system for Leishmaniasis canine, a
laboratory based project has been undertaken to identify
the antigens of L. infantum, by means of a immuno-
detection search of an expression library for genes of L.
infantum using dog serum with active visceral
Leishmaniasis.
It has been observed that most of the antigens
isolated by this method belong to the family of proteins
conserved during the course of evolution. The
identification of the B epitopes of these antigens
indicate, however, that in all cases the antigenic
determinants were localised in regions that were not well
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conserved.
In particular, the acidic ribosomal proteins LiP2a
and LiP2b are recognised by more than 80% of the VL
serums.
5 It has been confirmed that these proteins contain
disease specific antigenic determinants, and that the
recombinant proteins LiP2a and LiP2b, from which a
fragment had been removed, could be used as a specific
instrument able to distinguish between VL and Chages
disease.
It has also been shown that the PO ribosomal
protein of L. infantum, very highly conserved on the
evolutionary scale, is recognised by a high percentage of
VL dog serums. Furthermore, the antigenic determinants
are found exclusively on the C-terminus of the protein,
that is to say, in the region that has been poorly
conserved during the course of evolution.
It has been observed that in 78% of the VL dog
serums, antigens against H2A protein are also present,
and it has been confirmed that despite the sequence
identity in all the H2A proteins among eukaryotic
organisms, the humoral response to this protein in VL
serums is particularly provoked by determinants specific
to the Leishmania protein H2A.
The antigenic determinants recognised by the VL dog
serums are found at both termini of the H2A protein.
The obvious solution to the problem currently
encountered in this art would be to have an invention
that would allow the assembly of a synthetic chimeric
gene that contained the DNA regions encoding the
antigenic determinants specific to the proteins LiP2a,
LiP2b, LiPO, and H2A, with a view to constructing a
protein rich in antigenic determinants.
However, as far as the applicant is aware, there is
currently no invention that contains the characteristics
described as ideal, with a view to reaching the desired
aim. This aim is the construction of a protein rich in
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antigenic determinants, arising from the assembly of a
chimeric synthetic gene, that contains the DNA regions
encoding the antigenic determinants specific to the
aforementioned proteins.
DESCRIPTION OF THE INVENTION
In a first aspect, the invention relates to a
chimeric gene formed by the DNA sequences that encode
antigenic determinants of four proteins of L.infantum,
useful for the serum diagnosis of canine Leishmaniasis.
In a further aspect, the invention relates to a
protein encoded by said chimeric gene, containing one or
more of the antigenic determinants of four proteins of
L.infantum encoded by the chimeric gene.
The invention further relates to a diagnostic
method for determining the presence of canine
Leishmaniasis in a human being or an animal, in
particular a dog, and/or in samples of biological fluids
derived from humans or animals, such as a blood sample.
In this diagnostic method, the chimeric gene of the
invention or the protein encoded by it can be used.
Alternatively, in the diagnostic method,=a nucleic
acid probe sequences specific for the chimeric gene of
the invention, or a part thereof, can be used, i.e. to
establish the presence of canine Leishmaniasis in a
patient or a sample.
Also, in the diagnostic method, antibodies against
the protein encoded by the chimeric gene of the
invention, or a antigenic part thereof such as an
epitope, can be used.
The invention further relates to assays or other
qualitative or quantitative methods for determining the
presence of canine Leishmaniasis in a human being or an
,animal, in particular a dog, and/or in samples of
biological fluids derived from humans or animals, such as
a blood sample. Such assays can use the chimeric gene of
the invention, the protein encoded by it, probes specific
for the chimeric gene or part thereof, and/or antibodies
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directed to the protein encoded by the chimeric gene of
the invention, or any antigenic part thereof. Such assays
can further be carried out in a manner known per se, for
instance for probe-hybridization assays or immunoassays.
In a further aspect, the invention relates to
diagnostic kits, at least comprising either a chimeric
gene of the invention, a protein encoded by said chimeric
gene, a probe specific for the chimeric gene of the
invention, or an antibody directed to the protein of the
invention. The kits can further contain all components
for diagnostic kits and/or diagnostic assays known per
se.
It should be noted that when herein, reference is
made to the chimeric gene of the invention, this term
also encompasses nucleic acid sequences that can
hybridize with the sequence mentioned below under
moderate or stringent hybridizing conditions.
in this context, heterologous hybridisation
conditions can be as follows: hybridisation in 6 x SSC
(20xSSC per 1000 ml 175.3 g NaCl, 107.1 g sodium
citrate.5H2O, pH 7.0), 0.1% SDS, 0.05% sodium
pyrophosphate, 5* Denhardt's solution (100 x Denhardt's
solution per 500 ml : 10 g Fico11T"-400, 10 g polyvinyl-
pyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction
v)) and ,20 yg/ml denatured herring sperm DNA at 56 C for
18-24,hrs followed by two 30 min, washes in 5 x SSC, 0.1
% SDS at 56 C and two 30 min. washes in 2 x SSC, 0.1% SDS
at 56 C.
For instance, sequences that can hybridize with the
sequence mentioned below include mutant DNA sequences
which encode proteins with the same biological function
as the protein encoded by the sequence mentioned
hereinbelow. Such mutant sequences can comprise one or
more nucleotide deletions, substitutions and/or additions
to the sequence mentioned below. Preferably, the mutant
sequences still have at least 50%, more preferably at
least 70%, even more preferably more than 90 % nucleotide
I
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homology with the sequence given hereinbelow.
The term chimeric gene as used herein also
encompasses nucleic acid sequences that comprise one or
more parts of the sequence mentioned hereinbelow.
Preferably, such sequences comprise at least 10%, more
preferably at least 30%, more preferably at least 50% of
the nucleotide sequence given hereinbelow. Such sequences
may comprise a contiguous fragment of the sequence
mentioned hereinbelow, or two or more fragments of the
sequence given below that have been combined in and/or
incorporated into a single DNA sequence.
It should be noted that when herein, reference is
made to a protein encoded by the chimeric gene of the
invention, this term also includes mutant proteins that
still essentially have the same biological function.
Such mutant proteins can comprise one or more amimo acid
deletions, substitutions and/or additions compared to the
protein encoded by the sequence mentioned below.
Preferably, the mutant proteins still have at least 50%,
more preferably at least 70%, even more preferably more
than 90 % amino acid homology with the sequence given
hereinbelow.
The term protein also encompasses fragments of the
protein encoded by the chimeric gene of the invention.
Such fragments preferably still show the biological
activity of the full protein. Preferably, such proteins
comprise at least 30%, more preferably at least 50% of
the amino acid sequence of the full protein. Also, two or
more fragments of the full protein encoded by the
chimeric gene of the invention may be combined to form a
single protein.
Prpbes of the invention are such that they can -
most preferably selectively- hybridize with the chimeric
gene of the invention or part thereof, in particular
under moderate as stringent hybridizing conditions, such
.as those mentioned above. Preferably, a probe of the
invention will be essentially homologous with the
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nucleotide sequence of the chimeric gene of the invention
or a part thereof, i.e. show a homology of more than 80%,
preferably more than 90%, more preferably more than 95%.
A skilled person will be able to select suitable
probes. Usually, such probes will contain at least 15 bp,
preferably more than 24 bp, of the sequence given
hereinbelow.
The chimeric gene formed of the DNA sequences that
encode the antigenic determinants of four proteins of L.
infantum, useful for serological diagnosis of canine
Leishmaniasis and protein obtained, that the invention
proposes, in its own right constitutes an obvious novelty
within its field of application, as according to the
invention, a synthetic chimeric gene is produced that as
it is obtained by assembly, containing the DNA region
encoding the antigenic determinants specific to the
proteins LiP2a, LiP2b, LiPO and H2A, thus constructing a
protein rich in antigenic determinants. The chimeric gene
obtained is expressed in Escherichia coli and the product
has been analysed with respect to its antigenic
properties. The results confirm that this chimeric
protein maintains all the antigenic determinants of the
parent proteins and that it constitutes a relevant
diagnostic element for canine VL, with a sensibility that
oscillates between 80% to 93%, and a specificity of
between 96% to 100%.
More particularly, the chimeric gene formed by the
DNA sequences that encode the antigenic determinants of
four proteins of L. infantum, useful for the serological
diagnostic of canine Leishmaniasis and protein obtained
object of the invention, is produced by means of the
following stages, namely:
Construction of the chimeric gene. Methodology.
Cloning strategy.
Cloning of DNA sequences that encode antigenic
determinants of the histone protein H2A.
Cloning of the sequences that encode rLiP2a-Q and
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rLiP2b-Q.
Cloning of the sequence rLiPO-Q.
Cloning of the chimeric gene.
- Construction of the chimeric gene from the
5 construction of intermediate products.
Cloning of epitopes specific to the L. infantum
antigens.
- Construction of the final product
Construction of the chimeric gene that encodes a
10 polypeptide that contains all the selected antigenic
determinants.
- Evaluation of the final product.
Serums.
Purification of proteins
Electrophoresis of proteins and immuno-analysis.
Measurements by Fast-ELISA
- Evaluation of the final product.
Antigenic properties.
Sensitivity and specificity of the chimeric protein
CP in the serum diagnosis of canine VL.
The strategy followed by the cloning of DNA
sequences that encode each one of the selected antigenic
determinants is the same in all cases, and in a first
step, the sequence of interest is amplified by means of a
PCR and the use of specific oligonucleotides that contain
targets for restriction enzymes at the extremes.
For the cloning step, the amplified product is
directed by means of the appropriate restriction enzyme
and it is inserted in the corresponding restriction site
of the plasmid pUC18.
After sequencing the DNA, the insert is recovered
and sub-cloned to the corresponding restriction site of
the modified plasmid denominated pMAL-c2. The
modification is made by inserting a termination codon
downstream of the target Hindlil in the polylinker of
pMal-c2, denominating the resulting plasmid pMAL-c2*.
Regarding the cloning of the DNA sequence that
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encodes the antigenic determinants of the histone
protein H2A, it should be pointed out that the cDNA of
the clone cL71, that encodes the histone H2A of L.
infantum, is used as a template for the PCR reactions,
and for the DNA amplification, that encodes the N-
terminal region of the histone H2A, more exactly
rLiH2A-Nt-Q, the following oligonucleotides are used:
sense 5'- CCTTTAGCTACTCCTCGCAGCGCCAAG-3' (SEQ ID NO:1)
(position 84-104 of the sequence cL71); antisense 5'
CCTGGGGGCGCCAGAGGCACCGATGCG-3'(SEQ ID NO:2) (inverse
and complimentary to position 204-224 of the sequence
cL71).
The sequences that are included in the
oligonucleotides for the cloning and that are not
present in the parent sequence cL-71 are marked in
boldface type.
The amplified DNA fragment is cloned directly
from the restriction site XmnI of pMAI- c2*.
The fragment is sequenced by means of the
initiator #1234 malE and the antigenic C-terminal
region of histone H2A; in particular rLiH2A-Ct-Q, is
amplified with the following oligonucleotides. These
are:
Sense, 5'-GAATTCTCCGTAAGGCGGCCGCGCAG-3'(SEQ ID
NO: 3) (position 276-296 of the sequence cL71).
Antisense, 5'-GAATTCGGGCGCGCTCGGTGTCGCCTTGCC-3'
(SEQ ID NO:4) (inverse and complimentary to the
positions 456-476 of the plasmid cL71).
A triplet that encodes proline (indicated as
GGG after the underlined letters) is included in the
antisense oligonucleotide, the restriction site EccRI
that is included in both oligonucleotides for cloning
is indicated by underlining.
Regarding the cloning of the sequences that
encode rLiP2a-Q, it should be pointed out that the
regions of interest are amplified by PCR from cDNAs
encoding LiP2a and LiP2b.
The oligonucleotides that are used for
constructing the expression clone LiP2a-Q, are the
following.
Sense, 5'-GTCGACCCCATGCAGTACCTCGCCGCGTAC-3'
(SEQ ID NO:5).
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Anti-sense, 5'-GTCGACGGGGCCCATGTCATCATCGGCCTC-
3' (SEQ ID NO:6).
It should be pointed out that the Sall
restriction sites added to the 5' extremes of the
oligonucleotides have been underlined.
When constructing the expression clone LiP2b-Q,
the oligonucleotides used were:
Sense, 5'-TCTAGACCCGCCATGTCGTCGTCTTCCTCGCC-3'
(SEQ ID NO:7).
Anti-sense, TCTAGAGGGGCCATGTCGTCGTCGGCCTC-3'
(SEQ ID NO:8).
At the 5' extremes of the oligonucleotides the
restrictions sites are included for the enzyme XbaI
(underlined), and due to the cloning needs, an
additional triplet, encoding a proline residue, is
included downstream of the restriction site.
Regarding the cloning of the sequence rLiPO-Q,
it should be pointed out that the cloning of the DNA
sequence of the C-terminal region of the protein PO of
L. infantum is carried out by amplifying a clone of
cDNA called L27 and the following oligonucleotides:
Sense, 5'-CTGCAGCCCGCCGCTGCCGCGCCGGCCGCC-3'
(SEQ ID N0:9) (positions 1-24 of the L27 cDNA) and the
initiator of the pUC18 sequence (#1211), the amplified
DNA is directed by the enzymes PstI+HindIII, with later
insertion into the plasmid pMAL-c2.
The resulting clone is denominated pPQI and it
should be noted that the restriction site PstI is
included in the nucleotide with sense (underlined
sequence) and that the restriction target Hindlll is
present in the cDNA L27.
Regarding the cloning of the chimeric gene, it
should be pointed out that the DNA sequences that
encode the five antigenic determinants are assembled
into a chimeric gene, and this assembly is carried out
on the clone pPQI, to which the codifying regions for
the antigenic regions LiP2a-Q are added sequentially in
the 3' direction (naming the results of cloning pPQ2),
LiP2b-Q (clone pPQ3), LiH2a-Ct-Q (clone pPQ4) and
LiH2A-Nt-Q (clone pPQ5).
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Finally, the insert obtained after the SacI+HindIII
digestion of the final clone pPQ5 is inserted into the
pQE31'expression plasmid, naming the resulting clone pPQ.
DESCRIPTION OF THE DRAWINGS
To complete the description that is being made and
with the aim of aiding the understanding of the
characteristics of the invention, the present disclosure
is accompanied, as an integral part thereof, by a set of
plans of illustrative nature that are not limiting. The
following is represented:
Figure number 1. Expression, purification, and
antigenicity of the engineered Leishmania proteins. (A)
SDS-PAGE of E. coli lysates trasnfected with MBP-LiPO-
Ct-Q (lane 1), MBP-LiP2a-Q (lane 2), MBP-LiP2b-Q (lane
3), MBP-LiH2A-Ct-Q (lane 4), and MBP-LiH2A-Nt-Q (lane 5).
Mr: molecular . mass markers. (B)_ SDS-PAGE of the
corresponding recombinant proteins after purification
through the amylose column. (C) Western blot of the
reactivity of three pooled canine VL serum samples (final
dilution, 1:100) against the purified proteins. (D)
FAST-ELISA of the reactivities (means and SDs) of 26 VL
serum samples against the purified antigens. Dilution of
the sera 1:300.
Figure number 2. Sample of the different vectors
considered to obtain the chimeric gene object of the
invention, from which the pertinent protein destined to
carry., out an accurate diagnostic on animals or human
beings that show symptoms of Leishmaniasis will be
extracted.
Figure number 3. Corresponds to the identification
of the protein obtained from the chimeric gene, the
preparation of which is represented in Figure number 2.
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13a
Figure number 4. Expression, purification, and
antigenicity of all the intermediates and the final
chimeric protein. (A) SDS-PAGE of E. coli lysates
transformed with PQI (lane 1), PQII (lane 2), PQIII (lane
3), PQIV (lane 4), and PQV (lane 5). Mr: molecular mass.
(B) SDS-PAGE of the corresponding recombinant proteins
after purification through the amylose column. (C)
Western blot of the reactivity of three pooled canine VL
serum samples (final dilution, 1:100) against the
purified antigens. (D) SDS-PAGE of the pQE chimeric
expression product. Lane 1, lysates of E. coli harboring
the PQ protein; lane 2, the PQ protein after purification
through a Ni-nitrilotriacetic acid column. Mr: molecular
mass. (E) Western blot of the reactivity of a pool of
three canine VL serum samples against the purified PQ
protein. (F) FAST-ELISA of the reactivities (+ SDs) of 26
VL serum samples against each one of the intermediate
proteins PQI to PQIV and final products PQV and PQ.
Dilution of the sera 1:300 dilution.
Figure number 5. FAST-ELISA evaluation of the
diagnostic value of the recombinant PQ protein. Mean O.D.
values (plus SDs) of sera from dogs infected with L.
infantum (group 1; n 59), sera from dogs infected with
other pathogens or suffering from diseases other than
leishmaniasis (group 2; n 49), and sera from healthy
dogs (group 3; n 15). Dilucion of sera 1:300
35
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PREFERRED B 3ODIMHNT OF THE INVENTION
The chimeric gene formed from the DNA sequences
that encode the antigenic determinants of four proteins
of L. infantum, useful for the serological diagnosis of
canine Leishmaniasis and the protein obtained that is
being proposed are constituted from the construction of
intermediate products. In a first instance, cloning of
epitopes specific to the antigens of L. infantum is
carried out, which is configured on the basis of earlier
studies on the antigenic properties of four protein
antigens of L. infantum (LiP2a, PiPO, LiP2b, LiH2a),
which allow the existence of B epitopes to be defined for
these proteins, and which are specifically recognised by
the canine serums of VL.
With a view to improving the antigenic specificity
of these antigens with respect to the proteins of L.
infantum, the specific antigenic determinants are cloned
from these proteins. After deleting certain regions of
.20 these proteins these can be recognised by serums from
animals that are carriers of VL and other different
diseases.
By using the specific oligonucleotides and
amplification by PCR of regions specific to the genes
LiP2a, LiP2b, PO and H2A, several clones are constructed
that express the recombinant proteins rLiPO-Ct-Q, rLiP2a-
Q, rLiP2b-Q, rLiH2A-Ct-Q and rLiH2A-Nt-Q, just has been
detailed in the description of the invention relating to
the methodology., where the cloning details are described.
The recombinant proteins used are the following:
- rLiPO-Ct-Q, which corresponds to the 30 C-
terminal residues of the ribosomal protein LiPO.
rLiP2a-Q and rLiP2b-Q, that are derived from the
ribosomal proteins LiP2a and LiP2b respectively.
= Two sub-regions of the histone H2A, that
correspond to the 46 N-terminus residues (xLiH2A-Nt-Q),
and to the 67 C-terminus residues (residues (xLiH2A-Ct-
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,
15.
Q) .
Each one of the recombinant proteins fused to the
maltose binding protein (MBP) is expressed in B. Col.i, as
represented in Figure number 1, and they were purified by
affinity chromatography on a amylose column. After the
process of purification the electrophoresis was carried
out on the recombinant proteins (lanes 1 to 5) in Figure
number 3.
With the aim of analysing whether the recombinant
proteins were recognised by VL canine serums, a Western
blot was incubated, containing the recombinant proteins
in a mixture of three VL canine serums. Given that all
these proteins are recognised by the serums, it is
concluded that the antigenic determinants present in the
parent proteins are maintained in the recombinant
proteins.
The antigenic properties of the recombinant
proteins are compared with the antigenic determinants of
the parent antigens by means of a FAST ELISA, testing
against a collection of 26 VL canine serums, just as is
shown in the section of Figure number 1, and the fact
that the serums showed a similar reactivity value, both
against the selected antigenic regions and the
corresponding complete proteins, demonstrates that no
alteration to the antigenic epitope has occurred during
the cloning procedure.
In regard to the construction of the final product,
more exactly of the chimeric gene that encodes a
polypeptide that contains all the selected antigenic
determinants, it should be pointed out that the cloning
strategy, is indicated following Figure number 2 section
A. The intermediate products generated during the
process are shown.
A clone that expresses the proteins rLiPO-Ct-Q
(pPQI) .is used as the initial vector, and the fragments
of DNA that encode the proteins rLiPO-Ct-Q, rLiP2a-Q,
rLiP2b-Q, rLiH2A-Ct-Q and rLiH2A-Nt-Q are added
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sequentially using appropriate restriction sites.
After each cloning step, the correct orientation of
each one of the inserts is deduced from the size of the
expression products, and finally the complete nucleotide
sequence of the final clone pPQV is determined and the
amino acid sequence deduced from the sequence represented
in Figure number 3.
The polypeptide generated has a molecular mass of
38 kD, with an isoelectric point of 7.37, including
spacer sequences encoding proline, underlined in Figure
number 3. The aim of doing this is to efficiently
separate ' the antigenic domains and avoid possible
tertiary conformations that could interfere with the
stability and antigenicity of the final product.
The expression and recovery of each of the
intermediate products is shown in figure number 4, boxes
A and B. As was expected, after each addition, the size
of the expression product in the vector pMAL gradually
increases until reaching a molecular weight of 80 kDa.
Included in this are the sizes of the proteins rLiH2A-Ct
and rLiH2A-Ct, observing a certain degree of rupture
during purification.
The chimeric gene was also cloned in the plasmid
pQE, a vector that allows the expression of proteins with
a fragment of 6 histidines at the extreme N-terminus.
The resulting clone and the recombinant proteins
are denominated p?Q and PQ respectively.
The level of expression of the protein in bacteria
transformed with the pPQ plasmid and the purified
proteins are shown in Figure number 4, referred to in
particular with a D, with the protein PQ, purified by
affinity chromatography in denaturising conditions is
more stable that the recombinant protein pPQV represented
in Figure number 4, in box B.
In order to evaluate the final product a series of
materials were used, and obviously some techniques, as is
described below.
f
CA 02256125 1998-12-23
17
Serums of VL obtained from dogs of different
origins are used. The animals are evaluated clinically
and analytically in the pertinent laboratory, generally
in a Department of Parasitology, and all the positive
serums are assayed for indirect immuno-fluoresence (IIF).
The presence of amastigotes of the parasites of
these animals is confirmed by direct observation of the
popliteal and pleescapular lymph nodes, and a second
group of 33 serums of VL originating from other regions,
were given a positive diagnosis in the ELISA against
total protein extracts of the parasite and/or by IIF.
The serums of dogs affected by different diseases
that were not VL are obtained from different origins.
Within this group serums from the following infections
are found:
Mesocestoides spp.
Dyphylidium caninum
Uncinaria stenocephala
Toxocara canis
Dipetalonema dranunculoides
Demodex canis
Babesia canis
Ehrlichia cannis
Ricketsia ricketsiae.
The rest of the serums were obtained from dogs that
exhibited various clinical symptoms that were not related
to any infective process, and the serum controls were
obtained from fifteen carefully controlled healthy
animals.
Purification of the recombinant proteins expressed
by the clones pMA1-c2 is carried out by affinity
chromatography on amylose columns, and the purification
of the recombinant protein expressed by the clone pPQ was
performed on Ni-NTA resin columns in denaturising
conditions (Qiagen).
For analysing the proteins electrophoresis on 10%-
polyacrimide gels in the presence of SDS was carried out
CA 02256125 2008-06-27
18
under standard conditions. immunological analysis of the
proteins separated by electrophoresis was carried out on
nitrocellulose membranes to which the proteins had been
transferred. The transferred proteins were blocked with
dried 5% skimmed milk in a PBS buffer with 0.5% TweenT" 20.
The filters were sequentially brought into contact
with primary and secondary anti-serum in blocking
solutions and an immuno-conjugate labelled with
peroxidase was used as second antibody, visualising the
specific binding by means of an ECL system.
The Fast-ELISA was used instead of the classic
ELISA, and the sensitisation of the antigen was carried
out for 12 hours at room temperature.
The plates were sensitised with 100 pl of antigen
whose concentration in all cases was 2 g/ml.
After sensitising the wells the plates were
incubated for 1 hour with blocking solution (0.5%
powdered skimmed milk dissolved in PHS - 0.5% Tween- 20.
and the serums were diluted three hundred fold in
blocking solution).
The wells were incubated with serum for 2 hours at
room temperature, and after exposure to the antibody the
wells were washed with PBS-Tweenm 20.
Antibodies labelled with peroxidase were used as
second antibodies at a dilution of 1:2000 and the colour
of the reaction was developed using the substrate ortho-
phenylenediamine, measuring the absorption at 450 nm.
In regard to evaluation of the final product, it
should be pointed out that the antigenic properties were
determined by means of the pertinent study of the
reactivity of the VL canine serums against the chimeric
protein and against each one of the intermediate products
in a "Western blot" assay. All the intermediate products
maintained their antigenicity as well as did the final
pPQV prooduct, throughout the whole of the cloning
process.
CA 02256125 1998-12-23
19
It should also be pointed out that the recombinant
protein expressed by the pPQ plasmid was recognised by
the VL serums. With a view to analysing with greater
precision the antigenic properties of the chimeric
protein and the intermediate products, an analysis of the
reactivity of a wide variety of VL canine serums was
performed by means of a fast-ELISA against the
recombinant proteins, as is shown in the section F of
Figure number 4. It can be highlighted that the
absorption values and the sensitivity of the different
intermediate products of cloning increases after each
addition stop. It should also be pointed out that the
protein pQI is recognised by most of the VL serums and
the protein PQII equally by most of the serums. This
proportion is greater for the protein PQIII, and the
proteins PQIV, PQV and PQ are recognised by practically
all the serums.
According to what has been discussed above, the
percentage of recognition shown by the serums was similar
both in the case of assaying the chimeric proteins PQV
and PQ, and of assaying a mixture of recombinant proteins
rLiPO-Ct-Q, rLiP2a, rLiP2b and rLiH2A. It was seen that
the antigenic properties of each one of the 5 selected
antigenic regions are present in the PQ expression
product, and therefore this product can be used for
diagnosis instead of a mixture of the antigens expressed
individually.
With a view to determining whether the chimeric
protein can be used for canine VL serum diagnosis, and
according to the pertinent analysis of a wide variety of
canine serums against this protein, bearing in mind that
according to the clinical characteristics of the animals,
the canine serums have been classified into three groups.
A first group consisted of serum of dogs with a real L.
infantum infection. A second group was composed of serums
of dogs that had various clinical symptoms without being
infected with Leishmania, including dogs infected with
CA 02256125 2011-02-02
parasites different to Leishmania incorporated into this
group. The rest of the serums originated from dogs that
exhibit clinical symptoms that could be confused with
those observed during Leishmaniasis.
5 The third group was made up of control serums,
originated from serums of healthy dogs.
In figure number 5 the average values of reactivity
are shown for each group of serums, the reactivity of the
VL serums reaching an average reactivity value of 0.8
10 (S.D. = 0.4).
Within this group the reactivity of 12 serums is
less than 0.35, while the reactivity of 10 serums reaches
values of between 0.35 and 0.5. It is observed that the
reactivity over 23 serums varies between 0.5 and 1.0,
15 with 14 serums showing a reactivity greater than 1Ø
The average absorption value of the serums of the
second group, that is to say, the group in which animals
infected with Leishmania parasites and parasites
different to Leishmania parasites, is 0.2 (S.D. = 0.05)
20 and the reactivity of the control serums, that is to say,
the third group, is 0.1 (S.D. = 0.003).
The data presented above indicate that the chimeric
protein PQ in the FAST ELISA has a sensitivity of 80% for
the VL diagnosis, if the cut-off value is defined as the
average reactivity value of the serums of group 2 plus
three S.D.'s (that is to say 0.35).
The sensitivity of the assayed group reaches 93%,
if the cut-off value is defined by the reactivity values
of the control group, and the data indicate that the
protein CP has a specificity of 96% for the VL diagnosis,
when the cut-off value is defined by the aforementioned
serums of group 2, and only two serums from group 2
showed reactivity between 0.35 and 0.40. 100%
specificity in the assay was reached when the reactivity
values of healthy dogs were considered.
The process to be used is the followin.q:
1.- The microtitre plates are covered with
CA 02256125 2008-06-27
21
antibodies by incubated 100 Al of a solution that
contains 1 g/ml of antigen dissolved in a buffer PBS -
0.5% Tween 20 - 5% skimmed milk (Buffer A).
The incubation is performed for 12 hours at room
temperature, and then the plates are washed three times
with the same buffer containing no antigen. The dry
antigenated plates could be maintained' at room
temperature.
2.- A first incubation of the wells was carried out
with the serum of animal at a dilution of 1/200 in buffer
A. The incubation lasts for 1 hour.
3.- The wells are washed with buffer A, as
described in point 1, three times with a wash flask.
4.- They are incubated with a second antibody (IgG
labelled with peroxide) diluted 1:2000 in buffer A,
carrying out the incubation for 1 hour.
5.- The wells are washed once again with buffer A
three times, as was indicated in the third section, that
is to say with a wash flask.
6.- The reactivity is revealed using the substrate
ortho-phenylenediamine and the absorption measured at 450
nm.
The protein used for the diagnosis extracted from
the chimeric gene is identified as follows:
I MBP IEGRPLATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGA 50
SGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVV 104
PNISKAMAKKKGGKKGKATPSAPEFGDSSRPMSTKYLAAYALASLSKASPSQAD 158
VEAICKAVHIDVDQATLAFVMESVTGRDVATLTAEGAAKMSAMPAASSGAAAGV 212
TASAAGDAAPAAAAAKKDEPEEEADDDMGPSVRDPMQYLAAYALVALSGKTPSK 266
ADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAV 320
STAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESD 374
EDDFGMGGLF (SEQ ID NO:10)
It is not considered necessary to extend this
CA 02256125 1998-12-23
22
description in order that someone skilled in the art can
understand the scope of the invention and the advantages
that it confers.
The materials, form, size and disposition of the
elements are susceptible to change, provided it does not
suppose a change in the essence of the invention.
The terms in which this disclosure has been written
should always be considered as broad in nature and not
limiting.
CA 02256125 2011-03-31
23
SEQUENCE LISTING
(i) APPLICANT: C.B.F. Leti S.A.
(ii) TITLE OF INVENTION: Chimeric Gene Formed of the DNA
Sequences that Encode the Antigenic Determinants of Four
Proteins of L. Infantum, Useful for Serologic Diagnosis of
Canine Leishmaniasis and Protein Obtained
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BERESKIN & PARR
(B) STREET: 40 King Street West
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP: M5H 3Y2
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,256,125
(B) FILING DATE: 23-DEC-1998
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gravelle, Micheline
(B) REGISTRATION NUMBER: 4189
(C) REFERENCE/DOCKET NUMBER: 444-152
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311
(B) TELEFAX: (416) 361-1398
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCTTTAGCTA CTCCTCGCAG CGCCAAG 27
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
CA 02256125 2011-03-31
24
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCTGGGGGCG CCAGAGGCAC CGATGCG 27
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCTCCG TAAGGCGGCC GCGCAG 26
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAATTCGGGC GCGCTCGGTG TCGCCTTGCC 30
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GTCGACCCCA TGCAGTACCT CGCCGCGTAC 30
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02256125 2011-03-31
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GTCGACGGGG CCCATGTCAT CATCGGCCTC 30
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TCTAGACCCG CCATGTCGTC GTCTTCCTCG CC 32
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TCTAGAGGGG CCATGTCGTC GTCGGCCTC 29
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CTGCAGCCCG CCGCTGCCGC GCCGGCCGCC 30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02256125 2011-03-31
26
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ile Glu Gly Arg Pro Leu Ala Thr Pro Arg Ser Ala Lys Lys Ala Val
1 5 10 15
Arg Lys Ser Gly Ser Lys Ser Ala Lys Cys Gly Leu Ile Phe Pro Val
20 25 30
Gly Arg Val Gly Gly Met Met Arg Arg Gly Gln Tyr Ala Arg Arg Ile
35 40 45
Gly Ala Ser Gly Ala Pro Arg Ile Ser Glu Phe Ser Val Lys Ala Ala
50 55 60
Ala Gln Ser Gly Lys Lys Arg Cys Arg Leu Asn Pro Arg Thr Val Met
65 70 75 80
Leu Ala Ala Arg His Asp Asp Asp Ile Gly Thr Leu Leu Lys Asn Val
85 90 95
Thr Leu Ser His Ser Gly Val Val Pro Asn Ile Ser Lys Ala Met Ala
100 105 110
Lys Lys Lys Gly Gly Lys Lys Gly Lys Ala Thr Pro Ser Ala Pro Glu
115 120 125
Phe Gly Asp Ser Ser Arg Pro Met Ser Thr Lys Tyr Leu Ala Ala Tyr
130 135 140
Ala Leu Ala Ser Leu Ser Lys Ala Ser Pro Ser Gln Ala Asp Val Glu
145 150 155 160
Ala Ile Cys Lys Ala Val His Ile Asp Val Asp Gln Ala Thr Leu Ala
165 170 175
Phe Val Met Glu Ser Val Thr Gly Arg Asp Val Ala Thr Leu Thr Ala
180 185 190
Glu Gly Ala Ala Lys Met Ser Ala Met Pro Ala Ala Ser Ser Gly Ala
195 200 205
Ala Ala Gly Val Thr Ala Ser Ala Ala Gly Asp Ala Ala Pro Ala Ala
210 215 220
Ala Ala Ala Lys Lys Asp Glu Pro Glu Glu Glu Ala Asp Asp Asp Met
225 230 235 240
Gly Pro Ser Val Arg Asp Pro Met Gln Tyr Leu Ala Ala Tyr Ala Leu
245 250 255
Val Ala Leu Ser Gly Lys Thr Pro Ser Lys Ala Asp Val Gln Ala Val
260 265 270
Leu Lys Ala Ala Gly Val Ala Val Asp Ala Ser Arg Val Asp Ala Val
275 280 285
Phe Gln Glu Val Glu Gly Lys Ser Phe Asp Ala Leu Val Ala Glu Gly
290 295 300
CA 02256125 2011-03-31
27
Arg Thr Lys Leu Val Gly Ser Gly Ser Ala Ala Pro Ala Gly Ala Val
305 310 315 320
Ser Thr Ala Gly Ala Gly Ala Gly Ala Val Ala Glu Ala Lys Lys Glu
325 330 335
Glu Pro Glu Glu Glu Glu Ala Asp Asp Asp Met Gly Pro Val Asp Leu
340 345 350
Gln Pro Ala Ala Ala Ala Pro Ala Ala Pro Ser Ala Ala Ala Lys Glu
355 360 365
Glu Pro Glu Glu Ser Asp Glu Asp Asp Phe Gly Met Gly Gly Leu Phe
370 375 380
