EP0465602A1 - METHOD FOR DETECTION AND/OR IDENTIFICATION OF $i(LYSSAVIRUS) INFECTIONS, CLONING AND EXPRESSION OF GENES CODING FOR PEPTIDES AND/OR FRAGMENTS OF PEPTIDES OF MOKOLA $i(LYSSAVIRUS), VACCINE AGAINST THE MOKOLA VIRUS AND/OR THE FAMILY OF $i(LYSSAVIRUSES) AS WELL AS METHOD FOR OBTAINING SAID VACCINE VIA - Google Patents
METHOD FOR DETECTION AND/OR IDENTIFICATION OF $i(LYSSAVIRUS) INFECTIONS, CLONING AND EXPRESSION OF GENES CODING FOR PEPTIDES AND/OR FRAGMENTS OF PEPTIDES OF MOKOLA $i(LYSSAVIRUS), VACCINE AGAINST THE MOKOLA VIRUS AND/OR THE FAMILY OF $i(LYSSAVIRUSES) AS WELL AS METHOD FOR OBTAINING SAID VACCINE VIAInfo
- Publication number
- EP0465602A1 EP0465602A1 EP90907112A EP90907112A EP0465602A1 EP 0465602 A1 EP0465602 A1 EP 0465602A1 EP 90907112 A EP90907112 A EP 90907112A EP 90907112 A EP90907112 A EP 90907112A EP 0465602 A1 EP0465602 A1 EP 0465602A1
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- European Patent Office
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- leu
- ser
- val
- glu
- ile
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a method for detecting and identifying Lyssavirus infections, to the cloning and to the expression of genes coding for peptides and / or fragments of peptides of Lyssavirus Mokola, to a vaccine against the Mokola virus and / or all of the Lyssaviruses as well as its process for obtaining by genetic engineering.
- Lyssavirus strains isolated from bats in northern Europe and Spain are currently being classified. These viruses, although currently only isolated from bats, pose the problem of protecting the human population and other animal species, and in particular the problem of vaccine protection against these viruses.
- the Mokola virus is also of concern, since it has been made responsible in Africa for isolated cases of fatal rabiform encephalitis in many species, including humans, and especially for an epidemic in domestic Malawi carnivores, including a dog. who was vaccinated against rabies.
- a number of documents describe nucleotide sequences of the rabies virus and the vaccines derived therefrom.
- the French patent THE WISTAR INSTITUTE 2,515,685 proposes a complementary synthetic DNA which codes for the glycoprotein of the rabies virus of strain ERA, which is defined by its nucleotide sequence, its initiation codon ATG and its termination codon TGA, which is a copy of the mRNA of said glycoprotein and which is a single-stranded cDNA.
- fragments of polypeptides of this cDNA between two cleavage sites cannot be greater than 50 amino acids.
- the deduced amino acid sequence of the glycoprotein comprises 524 amino acids with a signal peptide of 19 non-polar amino acids preceding the amino-terminal lysine residue and cleaved in the mature protein.
- the cDNA of the rabies virus and the mRNA of the glycoprotein can be used to transform a bacterium in order to produce a polypeptide in sufficient quantities to carry out immunization.
- European Patent Application TRANSGENE 94 887 relates to a vector, such as a phage or a plasmid, for the expression of an antigenic protein of rabies, and more particularly the glycoprotein, which comprises at least one efficient DNA sequence which code for said protein and a promoter for the expression of this sequence in a bacterium, the coding effective DNA sequence possibly being a total effective DNA sequence or a partial sequence lying between two determined cleavage sites.
- This vector is used to transform or transfect - depending on whether it is a plasmid or a phage - a bacterium by culture from which the antigenic protein of the desired rabies is obtained, which is challenged negated by its amino acid sequence and which is used as an active component of a rabies vaccine.
- the claims of this Application essentially relate to the partial polynucleotide sequence 71-1 421 which codes for the nucleoprotein, to the partial sequence 1 514-2404 which codes for the protein Ml, to the partial sequence 2 496-3 102 which codes for protein M2, on the partial sequence 5 417-5 500 which codes for the N-terminal part of protein L.
- nucleotide sequences and their fragments and others suggested in this Application inserted into vectors, modify these vectors which, when introduced into an appropriate host cell, oblige it to transcribe and translate the DNA sequences. the above to produce the corresponding proteins which can then be isolated from cell extracts of said host cell.
- This Application also covers recombinant DNAs which contain one of the abovementioned inserts placed under the control of a promoter derived from the genome of the SV40 virus, which are vectors which can be used to transform eukaryotic cells (such as Vero cells) and produce proteins endowed with immunological properties, the promoter being able, more generally, to be a viral or eukaryotic promoter recognized by the polymerases of the selected cells and which additionally comprises suitable polyadenylation sites downstream of the insert.
- This Patent Application men Furthermore, one of the major advantages of the invention is to provide DNA sequences derived from the genomic RNA of the rabies virus which, unlike the genome itself, can be isolated under a form devoid of nucleoproteins.
- the cDNAs according to this Patent Application can be used as probes, for example for the detection of the presence in a biological fluid, of the rabies virus, by hybridization.
- N, M1, M2 polypeptides as defined in this Patent Application and the aforementioned Articles, by their peptide structures, can be used to produce corresponding antibodies which can themselves be used for the in vitro diagnosis of the presence of polypeptides viral in a biological fluid, the protein M1 being of very particular interest, in particular as an immunogenic composition in association with a vehicle used in the production of vaccines.
- the article in the name of O. POCH et al. published in Biochemistry, 1988, 70, 1019-1029 describes cDNA fragments of the genome of an avirulent strain (AVO1) of the rabies virus.
- AVO1 avirulent strain
- the 3,386 nucleotide sequence from the 3 'end covers the genes encoding the leader RNA, nucleoprotein N, phosphoprotein M1 and the matrix protein M2, as well as the intergenic regions.
- Comparison of the AVO1 sequence with that of other strains of the rabies virus reveals significant conservation both at the nucleotide and amino acid level.
- rabies genome Comparison of the rabies genome with those of other non-segmented negative single-stranded RNA viruses (rhabdovirus and paramyxovirus) indicates that the signals for initiation and stop of transcription, located at the ends of each gene coding for a protein, and the regions phosphoprotein and matrix proteins which could be involved in the transcription process maintain a similar overall structure.
- the Mokola virus is a so-called "related" rabies virus: rabies vaccines provide very little protection against infection by the Mokola virus.
- mice vaccinated with the three vaccines available on the market (PV strain (Pasteur); PM strain (Mérieux); Flury LEP strain (Behring)) do not withstand a test by the virus Mokola.
- their serum contains only few antibodies capable of neutralizing the Mokola virus in vitro, and their lymphocytes exhibit low cytotoxicity with respect to target cells infected with the Mokola virus.
- This hybridization method is used to detect rabid transcripts in the brain.
- 32 p-labeled cDNA probes from the genome of the rabies strain PV (serotype 1) are used to identify very small amounts of specific viral RNA.
- the purified viral RNA is obtained after phenolic extraction.
- the RNA is fixed on nylon membranes and hybrid with a pool of M13 inserts complementary to 200-400 nucleotides of each rabies gene and each mRNA.
- the labeled hybridized probes are detected by autoradiography. Hybridization was observed with brain samples from animals inoculated with vulpine strains of rabies virus (serotype 1). A positive response was obtained for amounts of total RNA of 80 ng.
- Detection of viral transcripts remained possible on brains taken a week after the animal's death. A total correlation is observed in comparison with other techniques such as the detection of rabies antigens by the use of a fluorescent anti-rabies antibody or the isolation of the virus on murine neuroblastome cells.
- the purpose of the present invention is to provide a specific vaccine against the Mokola virus obtained by expressing viral antigens mainly involved in the immune response as well as a polyvalent vaccine directed against all the Lyssavirus serotypes, obtaining by engineering
- the advantage of genetics is that it solves the problems of virus production and provide a specific diagnostic agent that is very sensitive to Lyssavirus infections.
- the subject of the present invention is a process for the rapid detection and / or identification of small amounts of Lyssavirus present in a biological sample, characterized in that said sample suitably treated to extract the viral RNA and / or the transcription products of the Lyssavirus possibly present is:
- the cDNA sequence is brought into contact with a pair of suitable Lyssavirus primers to amplify at least one fragment of said cDNA, one of said primers being different from that of step (1) and the other primer being identical to or different from that of step (1);
- the PCR method is notably described by SAIKI et al. in Science, 1988, 239, 487 as well as in European Patent Applications CETUS No 200 362 and 201 184.
- the method of the present invention unexpectedly allows rapid detection (less than 12 hours) of a Lyssavirus infection from a saliva or organ sample and then identify the Lyssavirus.
- the primers are chosen from the group which comprises the primers specific for a strain, for a serotype of Lyssavirus, the specific primers specific for a Lyssavirus serotype and the primers consisting of a conserved sequence of a gene common to all Lyssaviruses and hereinafter called polyvalent primers.
- the primers for diagnosis or detection of a Lyssavirus (Lyssavirus infection: response of the Yes / No type) and the primers for differential diagnosis or typing ( Lyssavirus infection: details on type).
- the primer is a 3 'rabies primer, located in position 1-18 of the rabies and Mokola genomes.
- the primer is a rabies primer M2, located in position 2901-2918 of the rabies genome.
- the primer is a rabies primer consisting of 23 nucleotides, located in position 4665-4687 of the rabies genome and in position 4675-4697 of the Mokola genome, hereinafter called primer G .
- the primer is a rabies primer consisting of 24 nucleotides, located in position 5520-5543 of the rabies genome and in position 5545-5568 of the Mokola genome, hereinafter called primer L .
- the primer is a Mokola or rabic primer consisting of 18 nucleotides located in position 587-605 of the rabies and Mokola genomes, hereinafter called the Na primer.
- the primer is a Mokola or rabic primer consisting of 16 localized nucleotides in position 1013-1029 of the rabies and Mokola genomes, below called primer Nb.
- the detection of the amplified nucleotide sequence is carried out by hybridization using a probe or a battery of probes suitable for the various Lyssaviruses.
- the detection of the amplified nucleotide sequence is carried out by cleavage of said sequence with at least one battery of suitable restriction enzymes followed by separation by electrophoresis of the fragments obtained.
- the battery of enzymes comprises BamH I, Hind II, Hind III, Pst I.
- the battery of enzymes comprises Rsa I, Taq I, BstX I, Fnu4H I.
- the rapid detection method, according to the invention of small amounts of Lyssavirus in a sample, has the advantage of making it possible to diagnose Lyssavirus infection on early infections or on saliva samples taken from domestic animals suspected of being responsible for human contamination, several days before their death, contrary to current practice; indeed, for example, the pair of primers, Na primer and Nb primer as defined in the invention, located on the gene N and not far apart from one another (400 bp) allows the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
- the method according to the invention allows identification and typing of the Lyssavirus involved in the infection to be detected.
- the pair of primers, rabies primer G and rabies primer L as defined above, associated with a battery of appropriate restriction enzymes makes it possible to type both the different strains of rabies virus, the virus Mokola, as strains of Lyssavirus isolated from bats; the two aforementioned primers are chosen so as to correspond to conserved zones in Lyssaviruses, one in the distal part of the G gene, the other in the proximal part of the L gene, to allow the amplification of the region which separates, around 860 nucleotides (pseudogen psi), on all rabies strains as well as on Mokola.
- the amplified band is detected either by hybridization with a more or less specific strain-specific probe, or by hydrolysis in the presence of more or less strain-specific restriction enzymes.
- a direct sequencing technique can be applied directly to the piece of agarose containing the amplified band.
- the present invention also relates to the specific sequences of the Mokola virus, which sequences have made it possible to highlight the conserved areas and the variable areas in the genus Lyssa. virus and by comparison between the rabies and Mokola genomes, to assess the variability of each genomic region and to develop the detection and / or identification method of Lyssavirus above.
- the nucleotide sequence of the cDNA of the genomic RNA of the Mokola virus is characterized in that it comprises approximately 12,000 nucleotides.
- Said genomic RNA cDNA sequence is further characterized in that the 3 'and 5' ends are complementary, in that the 12 nucleotides of the 5 'end are identical to those of the PV strain of rabies virus, and in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the proteins N, M1, M2, G and L.
- sequence of the cDNA of the genomic RNA of the Mokola virus comprises the sequence in nucleotides and the deduced sequence in amino acids as follows (I):
- Val-Ile-Val-Asp-Ile-Val-Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp- GTG ATC GTC GAT ATA GTT CGC ACT AAC GTT GAG GGG AAT TGG 430
- TTC CAT AAA AAC TTT GAA GGG GAG ATT AAG AGA ATG TTT GAG 892
- GAG GAG GCC AGA GTA GAG GCT TCG CTC GCT GAT GAC GGG ACT 1228
- AGAACAATTA CCGCAACGGT GCCCGCTTTC AGCACAATAC ATATAAGCTA 3241 ACCACTGGTT TGTCTTCCTA TTCAGGGTCG AGCGAAAACG 3291 Met-Asn-Ile-Pro-
- GCT GAC ATC TTA CCC TCT AAG GGA TGT CTG AAA GTC GGG CAA 4431
- TTATAAAGAG TTGCCTTCTA AAATGGGCAC TCTATAGAGC CTTCAATCTT 5193 TTTGAGGTGC GGCAATATTA GCTTGAAATA ACCTTAAGGT CTAATTTCTC 5243
- TCT TTC CTC TCA AGG GGC CCT CTT AAG GGG TAC TTA GGA TCT 8946
- the subject of the invention is also, fragments of said sequence coding for one of the peptides and / or for a peptide fragment of the Mokola virus as well as fragments of said non-coding sequence corresponding to a variable region of the Mokola genome, that is to say ie not conserved of a gene common to all Lyssaviruses.
- coding fragments includes inter alia:
- N corresponds to nucleotides 71-1420 of said cDNA sequence
- non-coding fragments includes inter alia a fragment which corresponds to nucleotides 4897-5442 of said sequence, which fragment corresponds to the psi rabies pseudogen.
- the subject of the present invention is also the sequence of the genomic RNA of the Mokola virus, characterized in that it comprises approximately 12,000 nucleotides, in that it is a non-segmented and non-polyadenylated negative single-stranded RNA, in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the nucleoprotein N, the phosphoprotein M1, the matrix protein M2, the gly co-protein G and protein polymerase L and in that said genome is always associated with nucleoprotein N.
- the present invention also relates to the transcripts of the Mokola virus, characterized in that they consist of 5 successive monocistronic fragments coding from the 3 ′ end for the proteins N, M1, M2, G and L, to know :
- the present invention also relates to cDNA clones of the genomic RNA of the Mokola virus, characterized in that they correspond to the entire genome, from the 3 ′ end to the 5 ′ end, namely :
- pMD10 a fragment which measures 4,150 nucleotides, hereinafter called pMD10 and corresponding to the sequence coding for RNA "leader" for nucleoprotein N, protein M1, protein M2 and a fragment of the sequence coding for protein G ;
- pMA10 a fragment which measures 2,850 nucleotides
- pMA10 a fragment which measures 2,850 nucleotides
- pM7 a fragment which measures 2,850 nucleotides
- pMB5 a fragment of approximately 3,300 nucleotides, called pMB5 and corresponding to a fragment of the sequence coding for the protein L;
- pM12 a fragment of approximately 2800 nucleotides, hereafter called pM12, corresponding to a fragment of the sequence coding for the protein L;
- pMR15a a fragment of approximately 700 nucleotides, hereinafter called pMR15a and corresponding to a fragment of the sequence coding for the protein L as well as at the 5 'non-transcribed end of the genome;
- fragments taken separately, each have an ability to hybridize specifically to an RNA fragment originating from the transcription or replication of the Mokola genome or to a cDNA fragment.
- the pM7 clone was deposited under the number 1-847 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
- the pMB5 clone was deposited under the number 1-848 dated March 22, 1989 with the National Collection of microorganism cultures held by the Pasteur Institute.
- the pM12 clone was deposited under the number 1-849 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
- the clone pMR15a was deposited under the number 1-850 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
- the present invention also relates to nucleotide probes, characterized in that they consist of a nucleotide sequence as defined above or a fragment thereof, labeled with the aid of a marker such as a radioactive isotope, a suitable enzyme or a fluorochrome.
- probes According to one embodiment of said probes, mention may be made of the sequence 4675-5568 or a fragment thereof and in particular the fragment 4897-5442 or their complementary strands.
- Such probes are specific for the Mokola virus.
- the present invention also relates to peptides or peptide fragments, characterized in that they are encoded by at least one fragment as defined above or a fragment portion or a combination of several fragments as defined above.
- said peptide is encoded by the nucleoprotein N gene and has an amino acid sequence which corresponds to formula II below:
- said peptide is coded by the gene for the protein M1 and has an amino acid sequence which corresponds to formula III below: Met-Ser-Lys-Asp-Leu-Val-His- Pro-Ser-Leu-Ile-Arg-Ala-Gly- Ile-Val-Glu-Leu-Glu-Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile- Asn-Arg-Thr-Ile- Glu-Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu- Pro-Leu-Tyr-Val-Asp-Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu- Arg- Ile-Glu-Asp-Lys-Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu-Glu-Arg-Asp-Glu-Gly-Ser-Ser-Glu-Glu-Asp-Glu-Asp
- said peptide is encoded by the protein M2 gene and has an amino acid composition which corresponds to formula IV below:
- said peptide is coded by the glycoprotein G gene and is characterized by an amino acid sequence which corresponds to formula V below:
- said peptide is coded by the L protein gene and has one of the following amino acid sequences:
- peptides and / or peptide fragments in accordance with the invention are obtained by synthesis.
- the present invention also relates to a vector, characterized in that it contains at least one nucleotide sequence or a portion of this sequence as defined above.
- the present invention also relates to a vector for the expression of a peptide or of a peptide fragment or of a combination of fragments of peptides of the Mokola virus, characterized in that it is obtained by homologous recombination between a baculovirus. of wild strain and an appropriate shuttle vector comprising at least minus:
- said shuttle vector comprises:
- the gene or a fragment of the gene for the glycoprotein G of the Mokola virus is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said glycoprotein, under the control of the promoter of the polyhedrin gene.
- said expression vector was deposited under the number 1-851 dated March 22, 1989 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
- the gene or a fragment of the nucleoprotein N gene is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said nucleoprotein, under the control of the gene promoter polyhedrin.
- the process of expression of at least one peptide, a fragment of a peptide or a combination of frag peptides of the Mokola virus implements an expression vector in accordance with the invention, in appropriate lepidopteran cells, in particular the caterpillar Spodoptera frugiperda, Sf9 in the presence of wild-type baculovirus, for obtaining recombinant baculoviruses expressing the desired polypeptide.
- recombinant baculoviruses will be based on the morphology of the ranges obtained. Indeed, the presence of polyhedrin gives the beaches of wild virus a refractive aspect in light microscopy when they are illuminated by epiluminescence. Conversely, the dull appearance of the recombinant virus ranges, due to the absence of crystal, makes it easy to distinguish them.
- the expression level can be extremely high, of the order of 1 to 10 mg per liter of culture;
- polyhedrin is a late gene expressing itself after viral replication and the formation of nucleocapsides have taken place, even allows proteins toxic for the multiplication of the virus to be expressed.
- the present invention also relates to a vaccine against the Mokola virus, for human and / or veterinary use, characterized in that it comprises at least one peptide and / or a peptide fragment in accordance with the invention, optionally associated with at least one pharmaceutically acceptable vehicle.
- a vaccine against the Mokola virus for human and / or veterinary use, characterized in that it comprises at least one peptide and / or a peptide fragment in accordance with the invention, optionally associated with at least one pharmaceutically acceptable vehicle.
- said vaccine comprises the glycoprotein G and / or a fragment thereof and / or the nucleoprotein N or a fragment thereof.
- the combination of these two viral peptides has the following advantages: they intervene at different levels of the immune response; in fact the glycoprotein G preferentially induces the formation of neutralizing antibodies whereas the nucleoprotein N intervenes especially in cell-mediated immunity.
- the present invention also relates to a polyvalent Lyssavirus vaccine, characterized in that it comprises at least one peptide and / or peptide fragment in accordance with the invention, optionally associated with at least one peptide and / or a peptide fragment of at least one other Lyssavirus serotype.
- Lyssaviruses of serotype 1 rabies virus
- Lyssaviruses of serotype 2 Lagos bat virus
- Lyssaviruses of serotype 4 Duvenhage virus
- Lyssaviruses isolated on European bats close to serotype 4.
- said peptides and / or peptide fragments are advantageously associated with an appropriate support and / or an acceptable adjuvant, in particular the conventional adjuvants of human and veterinary vaccines.
- the present invention also relates to monoclonal antibodies specific for the peptides and / or peptide fragments of the Mokola virus, characterized in that they result from the immunization of mammals, in particular rodents, and more particularly of mice, by peptides and / or peptide fragments in accordance with the invention.
- the present invention also relates to an immunological method for detecting anti antibodies peptides and / or peptide fragments of the Mokola virus, which consists in detecting anti-Mokola antibodies possibly present in a biological sample using a peptide or a peptide fragment in accordance with the invention, by bringing together said biological sample with said peptide (s) or peptide fragment (s), to which the anti-Mokola antibodies bind if such antibodies are present in the biological sample to be analyzed, the reading of the result being revealed by an appropriate means, in particular EIA , RIA, fluorescence.
- an appropriate means in particular EIA , RIA, fluorescence.
- said peptides or peptide fragments are fixed on an appropriate solid support.
- Said immunological method can advantageously be of direct type, of indirect type or implement a sandwich or bi-site method.
- the revelation is carried out by means of a second antibody directed against the antibody to be assayed and appropriately labeled.
- This process allows the serum antibody titration and in particular makes it possible to verify the seroconversion of vaccinated individuals or to carry out serological surveys for epidemiological purposes.
- the subject of the present invention is also a method of rapid and specific detection of the Mokola virus which consists in detecting a Mokola virus, possibly present in a biological sample using at least one nucleotide probe according to the invention, by putting in the presence of said biological sample treated appropriately with said at least one nucleotide probe, to which / to which the genomic RNA and / or the transcripts of the Mokola virus binds, if such pro duits are present in the sample, the reading of the result being revealed by an appropriate means.
- the subject of the present invention is also a ready-to-use kit for implementing the method according to the invention for detecting and / or identifying at least one Lyssavirus, characterized in that it further comprises useful quantities of buffers and reagents suitable for carrying out said detection, appropriate doses of at least two suitable primers, appropriate doses of at least one nucleotide probe and appropriate doses of at least one enzyme restriction.
- the subject of the present invention is also a ready-to-use kit for implementing the method for determining in a biological sample, anti-Mokola antibodies, characterized in that it comprises at least:
- the peptide is glycoprotein G or a fragment thereof, fixed on an appropriate solid support.
- the peptide is nucleoprotein N or a fragment thereof, fixed on an appropriate solid support.
- This kit can also comprise appropriate doses of a second antibody directed against the antibody to be assayed labeled with the aid of an enzyme, a fluorescent substance or a radioactive isotope.
- the present invention further relates to a ready-to-use kit for implementing the method for detecting a Mokola virus according to the invention, characterized in that it comprises at least: - appropriate doses of at least one probe and / or fragment of nucleotide probe according to the invention; and
- the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the invention.
- Example 1 Cloning and sequencing of DNA complementary to the genomic RNA of the Mokola virus.
- the Mokola strain studied was isolated from a cat in Moscow.
- the virions are purified from the lysis plaques obtained on a culture of CER cells.
- the Mokola virus thus selected is cultured on BHK-21 cells and purified according to the method described by WIKTOR et al. (J. Virol., 1977, 21, 626-635) modified.
- the purified virions are incubated with 100 ⁇ g / ml of proteinase K (Merck) in 1.5% SDS, 100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA for 30 min at 37 ° C, followed by two phenol-chloroform extractions (vol / vol) and precipitation with ethanol.
- proteinase K Merck
- the first strand of cDNA is synthesized in the presence of 1 ⁇ g of genomic RNA using a molar ratio 10 times greater in the presence of one octodecameric primer at a time (the 3 'primer, located in position 1-18 or the primer M2, located in position 2,901-2,918 on the rabies genome, for example) in the presence of 50 units of reverse transcriptase, in a buffer containing 50 mM of Tris HCl pH 8.3, 8 mM of MgCl 2 , 100 mM KCl, 0.8 mM dATP, 0.8 mM dGTP, 0.3 mM dCTP, 0.3 mM dTTP, 0.2 ⁇ M 32 P dCTP, 0.2 ⁇ M 32 P dTTP, 30 U / ⁇ l RNasin for 2 hours at 42 ° C.
- the double stranded cDNA is prepared according to the GUBLER and HOFFMAN method and separated according to its size on a Biogel A-50 m column.
- fractions (approximately 15 to 20 ng of cDNA) corresponding to the largest cDNAs are inserted at the Pst I site of the plasmid pBR322 using the dC / dG extension method.
- the cDNA library obtained with the 3 'primer makes it possible to select the clone pMA10 with Mokola probes originating from the first cloning. From this, the pMD10 clone is selected.
- the inserts pMR15a and pMD10 have been sequenced and reach the 5 'and 3' ends of the genomic RNA respectively, proving that the five clones mentioned above are sufficient to cover the entire genome of the Mokola virus.
- Figure 2 compares the 3 'and 5' ends of the genome of the Mokola virus with those of the PV strain of the rabies genome.
- the complementary nucleotides between the two ends are specified using long vertical lines.
- the consensus sequences specifying the start of the N gene mRNA, the stop of the L gene mRNA and the start signal of the N protein are indicated.
- the nucleotides of the 3 'and 5' genomic ends of the Mokola virus which are identical to those of the PV strain are specified using short vertical lines. The residues are numbered from the genomic ends.
- FIG. 3 shows the heterologous and homologous hybridization of rabies probes located on the genes
- cytoplasmic RNA obtained from BHK-21 cells, is collected 6, 12, 24 or 48 hours after infection with the Mokola virus (traces 1) or infection with the rabies virus (trace 2); trace 3 corresponding to a negative control (uninfected cells), then is subjected to electrophoresis on 1.2% agarose gel - formaldehyde. The separated RNAs are deposited on nylon and hybrid membranes with a probe labeled with
- 32 P consisting either of the sequence 377-1039 of the cDNA of the rabies genome (probe N: A), or of the sequence
- the cDNA probes are selected in accordance with FIG. 1 to characterize the transcripts in vivo during an infection with the Mokola virus of BHK-21 cells.
- Six different transcripts are observed by the Northern blot analysis, as visible in FIG. 4 which shows the mRNAs of the Mokola virus hybridizing with the cDNAs N, N + M1, M1 + M2, G and L of the genome of the Mokola virus .
- the total cytoplasmic RNA of Mokola virus obtained from infected BHK-21 cells, is denatured in the presence of 50% formamide and 15 ⁇ g samples are subjected to electrophoresis on 1% agarose gel containing formaldehyde. The separated RNA is deposited on nylon membranes with cDNA probes labeled with 32 P (N, M1 + M2, N + M1, G and L). The arrows indicate the positions of the 18 S and 28 S ribosomal RNA, revealed by staining with ethidium bromide and the identity of the different mRNAs is revealed by autoradiography.
- Example 3 Method for determining the glycoprotein G of Mokola virus in a preparation or a biological liquid in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated with horseradish peroxidase.
- the suspension is clarified by centrifugation at 1500 xg for 30 min at 4 ° C.
- the clarified supernatants from each sample are distributed in duplicate in the wells of microtitration plates sensitized with anti-glycoprotein G antibodies in accordance with the invention (200 ⁇ l / well).
- the microplates are then incubated for one hour at 37 ° C. After repeated washing with PBS-Tween buffer, each well receives 200 ⁇ l of anti-glycoprotein G antibody conjugated with horseradish peroxidase.
- the microplates are then incubated for one hour at 37 ° C, then washed again.
- the viral antigen is then quantified by the appearance of a coloration, when the substrate of the peroxidase is added; the substrate is a mixture of o-phenylenediamine and hydrogen peroxide (200 ⁇ l / well).
- the microplates are left 20 min at room temperature to allow the staining to develop.
- the reaction is stopped by adding H 2 SO 4 N (50 ⁇ l / well).
- the coloration is measured with a spectrophotometer.
- Example 4 Method for determining the glycoprotein G of Mokola virus in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated to fluorescein isothiocyanate.
- Cell cultures infected with the Mokola virus are fixed for 30 min in cold acetone. Staining is carried out by covering the slides for 30 min at 37 ° C. with anti-glycoprotein G antibodies of Mokola virus conjugated to fluorescein isothiocyanate. Evans blue (1/5000) is used as a counter-dye. The slides are washed by immersion in PBS at pH 7.6 for 5 min. A mounting medium at the glycerin is used and the slides are examined under an epifluorescence microscope.
- Example 5 Method for detecting and titrating anti-glycoprotein 6 antibodies of the Mokola virus, in the blood of vaccinated individuals, with peptides and / or fragments of peptides in accordance with the invention.
- This test is based on the use of a solid phase, on which the virus glycoprotein is attached, and of an enzyme conjugate, protein A of Staphylococeus aureus coupled with peroxidase.
- Two-fold dilutions are made of positive and negative control sera. Unknown sera or plasmas are diluted to 1/100. 100 ⁇ l of these different sera are deposited in each well.
- the adhesive film is removed; the contents of each plate are aspirated, three successive washings of the wells are carried out, then the plates are dried.
- the contents of the wells are aspirated, washed four times and then the plates are dried.
- 3.5 ⁇ l (approximately 0.5 picomoles) of cloned M13 matrix are placed in the presence of 1 ⁇ l (0.5 picomoles) of universal primer and 0.5 ⁇ l of a buffer comprising 10 mM Tris-HCl pH 7, 5, 10mM MgCl 2 , 50mM NaCl, ImM DTT.
- the mixture hermetically closed, is immersed in a bain-marie which is brought to a boil and then allowed to cool gradually on the bench. After 1/2 h, the water is at 40 ° C and the hybridization is done.
- reaction takes place for 20 min at + 37 ° C, then is stopped by heating for 3 min at + 65 ° C.
- the M13 matrix, copied by the polymerase from the universal primer is in double strand over a length depending on the amount of mononucleotides initially introduced into the medium. In addition, this length varies statistically from one clone to another.
- the synthetic reaction mixture (15 ⁇ l) is brought to a salt concentration suitable for the restriction enzyme chosen (MANIATIS et al., 1982), and the cut takes place for 1 h, at the appropriate temperature, in a final volume of 20 ⁇ l.
- An identical volume of a denaturing deposit buffer is then added. After thermal denaturation, the mixture is deposited on a 6% denaturing acrylamide-urea gel (0.5 mm thick). The migration lasts 1 hour at 1200 volts.
- the radioactive probe is identified by autoradiography and then cut out.
- This probe can then be used for direct hybridization on blots or for S1 nuclease mapping.
- the techniques we follow are classic, although slightly modified. Here are some details:
- Example 7 Detection of a Mokola virus RNA using a probe in accordance with the invention (RNA blots)
- a hybridization medium which includes:
- BSA bovine serum albumin
- Example 8 Method for rapid identification (typing) of small quantities of Lyssavirus.
- RNA from BHK21 fibroblastic cells or N2A murine neuronal cells infected with a Lyssavirus (classic Rabies strains Pasteur, PV, CVS, Av01, PM, ERA, wild rabies strains "Gabon dog", “Brazilian bat “,” European fox “, Mokola, etc ).
- primer G ", corresponding to an oligonucleotide long 23 nucleotides, of direction (+) and extending between positions 4665 and 4687 of the rage PV genome, and a DNA of sense (+) complementary to the genome is specifically initiated and synthesized as described in Example 1.
- the reaction medium is extracted with phenol, precipitated with ethanol, washed twice with 70% ethanol, then dried. It is then taken up in 50 ⁇ l comprising:
- the primer "L” corresponds to an oligonucleotide 24 nucleotides long, of direction (-) and extending between positions 5520 and 5543 of the PV rabies genome.
- the primer G hybridizes with the region 4675-4697 of the nucleotide sequence (I) above, which primer G has a homology of the order of 80% with the region 4675-4697 of the sequence (I) of the Mokola virus.
- Primer L hybridizes specifically with region 5545-5568 of the nucleotide sequence of formula (I) of the Mokola virus.
- reaction medium is placed in a 1.5 ml eppendorf tube, covered with 100 ⁇ l of mineral oil to avoid evaporation, and incubated in a "P.C.R. machine" subjecting him to the following stages:
- This step is carried out once, then it is repeated 40 times in succession, limiting the denaturation time to 2 min.
- the amplified cDNA band can be observed after migration of the reaction products on an appropriate agarose gel. After transfer, it can be characterized by means of an appropriate probe, originating from the cloning of the genome of the strain analyzed or of another strain of Lyssavirus.
- Figure 7 corresponds to an electrophoresis gel and has the following 16 traces:
- the amplified cDNA band can also be hydrolyzed by selected restriction enzymes capable of quickly differentiating:
- the other two enzymes are more group specific:
- BstX I only cuts PV, ERA and SAD; .
- Fnu4H I only cuts wild rabies strains and the Mokola virus.
- FIG. 8 shows that the amplified bands originating from the ERA and CVS strains are respectively and specifically cut by Bstx I and BamH I.
- Example 9 Method for rapid detection (Yes / No response) of small amounts of Lyssavirus.
- the primers Na and Nb as defined in the invention allow, for example, the amplification of the 587-1029 region of the nucleotide sequence of formula (I) of the Mokola virus which is a conserved region of a Lyssavirus; this method of rapid detection of small amounts of Lyssavirus in a sample in accordance with the invention has the advantage of making it possible to make a diagnosis of infection with Lyssavirus on early infections or on saliva samples taken from suspect domestic animals human contamination, several days before their death, contrary to current practice; indeed, the primers Na and Nb as defined in the invention, located on the N gene and not far apart from one another (400 bp) allow the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
- the inserts pMD10 and pMA10 derived from the cloning of the Mokola genome, each cover half of the glycoprotein G gene. Thanks to the restriction site BglI which they have in common, they can be joined in a single insert pM7 covering the 6,830 nucleotides 3 'of the genome. from this, we isolate the cDNA from the glyco gene protein G by a double digestion combining the restriction enzymes FnuDII and Sspl, which is inserted between the promoter region 5 ′ of the baculovirus polyhedrin gene and its region 3 ′ corresponding in particular to the polyadenylation signal. This construction is carried out in a vector pUC, to allow its multiplication.
- Figure 5b corresponds to a negative control.
- the glycoprotein was also clearly localized on the surface of the cells infected with the recombinant virus, by means of fluorescent monoclonal antibodies specific for the glycoprotein of the Mokola virus (FIG. 6).
- traces 1, 2 and 3 correspond to Spodoptera frugiperda cells after infection with the recombinant baculovirus expressing the glycoprotein G of the Mokola virus: the arrow shows the location of the glycoprotein G on the gel; Trace T corresponds to a negative control.
- Example 11 Demonstration of the immunogenic power of the glycoprotein of the Mokola virus (G. MOK).
- FIG. 9 shows that the mortality curves of unvaccinated (NV) mice and vaccinated with SF9 cells infected with NPEV del are little different. SF9 cells infected with NPEV del therefore do not give any non-specific protection to mice.
- FIG. 10 shows that a vaccine dose of 100,000 SF9-G.MOK cells is sufficient to protect mice and 10 6 SF9-G.MOK cells are sufficient to obtain a protection index greater than 2.5.
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Abstract
Procédé de détection et d'identification des infections à Lyssavirus, clonage et expression de gènes codant pour des peptides et/ou pour des fragments de peptides du Lyssavirus Mokola, vaccin contre le virus Mokola et/ou l'ensemble des Lyssavirus ainsi que son procédé d'obtention par génie génétique. Le procédé de détection et/ou d'identification rapide de faibles quantités de Lyssavirus présents dans un échantillon biologique convenablement traité pour extraire l'ARN viral et/ou les produits de transcription des Lyssavirus éventuellement présents comprend: (1) la mise en contact dudit échantillon avec au moins une amorce appropriée des Lyssavirus, pour obtenir l'ADNc de l'ARN génomique ou de l'ARNm; (2) puis la séquence d'ADNc est mise en contact avec une paire d'amorces appropriées des Lyssavirus pour amplifier au moins un fragment dudit ADNc, l'une desdites amorces étant différente de celle de l'étape (1) et l'autre amorce étant identique à ou différente de celle de l'étape (1); (3) après quoi, la séquence d'ADNc amplifiée est détectée par un moyen approprié.Method for detecting and identifying Lyssavirus infections, cloning and expression of genes encoding peptides and / or fragments of peptides of Lyssavirus Mokola, vaccine against Mokola virus and / or all Lyssaviruses as well as its method obtained by genetic engineering. The method of rapidly detecting and / or identifying small amounts of Lyssavirus present in a biological sample suitably processed to extract viral RNA and / or transcription products of Lyssaviruses possibly present comprises: (1) contacting said sample with at least one suitable primer of Lyssaviruses, to obtain the cDNA of the genomic RNA or of the mRNA; (2) then the cDNA sequence is brought into contact with a pair of suitable primers of Lyssaviruses to amplify at least one fragment of said cDNA, one of said primers being different from that of step (1) and the another primer being identical to or different from that of step (1); (3) after which, the amplified cDNA sequence is detected by an appropriate means.
Description
PROCEDE DE DETECTION ET/OU D'IDENTIFICATION DES INFECTIONS A LYSSAVIRUS, CLONAGE ET EXPRESSION DE GENES CODANT POUR DES PEPTIDES ET/OU DES FRAGMENTS DE PEPTIDES DU LYSSAVIRUS MOKOLA, VACCIN CONTRE LE VIRUS MOKOLA ET/OU L'ENSEMBLE DES LYSSAVIRUS AINSI QUE PROCEDE D'OBTENTION DUDIT VACCIN PAR GENIE GENETIQUE. METHOD FOR THE DETECTION AND / OR IDENTIFICATION OF LYSSAVIRUS INFECTIONS, CLONING AND EXPRESSION OF GENES ENCODING PEPTIDES AND / OR FRAGMENTS OF LYSSAVIRUS MOKOLA PEPTIDES, VACCINE AGAINST MOKOLA VIRUS AND / OR THE WHOLE LYSSAVUS OBTAINING THE VACCINE BY GENETIC ENGINEERING.
La présente invention est relative à un procédé de détection et d'identification des infections à Lyssavirus, au clonage et à l' expression de gènes codant pour des peptides et/ou des fragments de peptides du Lyssavirus Mokola, à un vaccin contre le virus Mokola et/ou l'ensemble des Lyssavirus ainsi qu'à son procédé d'obtention par génie génétique. The present invention relates to a method for detecting and identifying Lyssavirus infections, to the cloning and to the expression of genes coding for peptides and / or fragments of peptides of Lyssavirus Mokola, to a vaccine against the Mokola virus and / or all of the Lyssaviruses as well as its process for obtaining by genetic engineering.
Au cours des vingt dernières années, ont été isolés de part le Monde des Rhabdovirus apparentés au virus rabique, dont la classification a été établie sur la base d'expériences de séroneutralisation croisée et de fixation du complément. Ainsi, le genre Lyssavirus s'est vu séparé en quatre sérotypes différents représentés respectivement pas le virus rabique (serotype 1), le virus Lagos bat (serotype 2 ; Afrique), le virus Mokola (serotype 3 ; Afrique) et le virus Duvenhage (serotype 4 ; Afrique du Sud). Les souches de Lyssavirus isolées des chauve-souris d'Europe du Nord et d'Espagne sont actuellement en cours de classement. Ces virus bien qu'actuellement uniquement isolés de chauve-souris posent le problème de la protection de la population humaine et des autres espèces animales, et notamment le problème de la protection vaccinale contre ces virus. Le virus Mokola est également préoccupant, puisqu'il a été rendu responsable en Afrique de cas isolés d'encéphalites rabiformes fatales chez de nombreuses espèces, dont l'Homme, et surtout d'une épidémie chez des carnivores domestiques du Zimbabwe, dont un chien qui était pourtant vacciné contre la rage.
Un certain nombre de documents décrivent des séquences nucléotidiques du virus rabique et des vaccins en découlant. Over the past twenty years, the World of Rhabdoviruses related to the rabies virus have been isolated from the world, their classification being established on the basis of experiences of cross-neutralization and complement fixation. Thus, the genus Lyssavirus has been separated into four different serotypes represented respectively by the rabies virus (serotype 1), the Lagos bat virus (serotype 2; Africa), the Mokola virus (serotype 3; Africa) and the Duvenhage virus ( serotype 4; South Africa). Lyssavirus strains isolated from bats in northern Europe and Spain are currently being classified. These viruses, although currently only isolated from bats, pose the problem of protecting the human population and other animal species, and in particular the problem of vaccine protection against these viruses. The Mokola virus is also of concern, since it has been made responsible in Africa for isolated cases of fatal rabiform encephalitis in many species, including humans, and especially for an epidemic in domestic Zimbabwe carnivores, including a dog. who was vaccinated against rabies. A number of documents describe nucleotide sequences of the rabies virus and the vaccines derived therefrom.
Le Brevet français THE WISTAR INSTITUTE 2 515 685 propose un ADN synthétique complémentaire qui code pour la glycoprotéine du virus rabique de souche ERA, qui est défini par sa séquence en nucléotides, son codon d'initiation ATG et son codon de terminaison TGA, qui est une copie de l'ARNm de ladite glycoprotéine et qui est un ADNc monocaténaire. The French patent THE WISTAR INSTITUTE 2,515,685 proposes a complementary synthetic DNA which codes for the glycoprotein of the rabies virus of strain ERA, which is defined by its nucleotide sequence, its initiation codon ATG and its termination codon TGA, which is a copy of the mRNA of said glycoprotein and which is a single-stranded cDNA.
Les fragments de polypeptides de cet ADNc entre deux sites de coupure ne peuvent pas être supérieurs à 50 acides aminés. The fragments of polypeptides of this cDNA between two cleavage sites cannot be greater than 50 amino acids.
La séquence déduite en acides aminés de la glycoprotéine comprend 524 acides aminés avec un peptidesignal de 19 acides aminés non-polaires précédant le résidu lysine amino-terminal et clivé dans la protéine mature. The deduced amino acid sequence of the glycoprotein comprises 524 amino acids with a signal peptide of 19 non-polar amino acids preceding the amino-terminal lysine residue and cleaved in the mature protein.
Son poids moléculaire est d'environ 67 000. L'ADNc du virus rabique et l'ARNm de la glycoprotéine peuvent être utilisés pour transformer une bactérie dans le but de produire un polypeptide en quantités suffisantes pour réaliser une immunisation. Its molecular weight is about 67,000. The cDNA of the rabies virus and the mRNA of the glycoprotein can be used to transform a bacterium in order to produce a polypeptide in sufficient quantities to carry out immunization.
La Demande de Brevet européen TRANSGENE 94 887 concerne un vecteur, tel qu'un phage ou un plasmide, d'expression d'une protéine antigénique de la rage, et plus particulièrement la glycoprotéine, qui comporte au moins une séquence d'ADN efficace qui code pour ladite protéine et un promoteur de l'expression de cette séquence dans une bactérie, la séquence d'ADN efficace codante pouvant être une séquence d'ADN efficace totale ou une séquence partielle comprise entre deux sites de coupures déterminés. Ce vecteur est utilisé pour transformer ou transfecter -selon qu'il s'agit d'un plasmide ou d'un phage- une bactérie par culture de laquelle on obtient la protéine antigénique de la rage recherchée, qui est défi
nie par sa séquence en acides aminés et qui est utilisée comme constituant actif d'un vaccin antirabique. European Patent Application TRANSGENE 94 887 relates to a vector, such as a phage or a plasmid, for the expression of an antigenic protein of rabies, and more particularly the glycoprotein, which comprises at least one efficient DNA sequence which code for said protein and a promoter for the expression of this sequence in a bacterium, the coding effective DNA sequence possibly being a total effective DNA sequence or a partial sequence lying between two determined cleavage sites. This vector is used to transform or transfect - depending on whether it is a plasmid or a phage - a bacterium by culture from which the antigenic protein of the desired rabies is obtained, which is challenged negated by its amino acid sequence and which is used as an active component of a rabies vaccine.
La Demande de Brevet européen INSTITUT PASTEUR et CNRS 237 686 du 18 mars 1986 -qui reproduit les données essentielles d'un Article paru dans Nucleic Acids Res., 1986, 14, 6, 2671-2683 et d'un Article paru dans Proc. Natl. Acad. Sci. USA, 1986, 83, 3914-3918 -donne la séquence partielle (jusqu'en position 5500) de l'ARN monocaténaire négatif non segmenté du virus rabique qu'elle revendique, tout comme l'ADNc qui en est dérivé. The European Patent Application INSTITUT PASTEUR and CNRS 237 686 of March 18, 1986 - which reproduces the essential data of an Article published in Nucleic Acids Res., 1986, 14, 6, 2671-2683 and an Article published in Proc. Natl. Acad. Sci. USA, 1986, 83, 3914-3918 gives the partial sequence (up to position 5500) of the unsegmented negative single-stranded RNA of the rabies virus which it claims, as well as the cDNA which is derived therefrom.
Cependant les revendications de cette Demande portent essentiellement sur la séquence polynucléotidique partielle 71-1 421 qui code pour la nucléoprotéine, sur la séquence partielle 1 514- 2 404 qui code pour la protéine Ml, sur la séquence partielle 2 496-3 102 qui code pour la protéine M2, sur la séquence partielle 5 417-5 500 qui code pour la partie N-terminale de la protéine L. However, the claims of this Application essentially relate to the partial polynucleotide sequence 71-1 421 which codes for the nucleoprotein, to the partial sequence 1 514-2404 which codes for the protein Ml, to the partial sequence 2 496-3 102 which codes for protein M2, on the partial sequence 5 417-5 500 which codes for the N-terminal part of protein L.
Ces séquences nucléotidiques et leurs fragments et d'autres encore suggérées dans cette Demande, insérées dans des vecteurs, modifient ces derniers qui, lorsqu'ils sont introduits dans une cellule-hôte appropriée, l'obligent à transcrire et traduire les séquences d'ADN susdites pour produire les protéines correspondantes qui peuvent alors être isolées des extraits cellulaires de ladite cellule-hôte. These nucleotide sequences and their fragments and others suggested in this Application, inserted into vectors, modify these vectors which, when introduced into an appropriate host cell, oblige it to transcribe and translate the DNA sequences. the above to produce the corresponding proteins which can then be isolated from cell extracts of said host cell.
Cette Demande couvre également les ADN recombinants qui contiennent l'un des inserts précités placés sous le contrôle d'un promoteur dérivé du génome du virus SV40, qui sont des vecteurs pouvant être utilises pour transformer des cellules eucaryotes (telles que les cellules Vero) et produire des protéines douées de propriétés immunologiques, le promoteur pouvant, de façon plus générale, être un promoteur viral ou eucaryote reconnu par les polymérases des cellules choisies et qui comporte en outre des sites de polyadénylation convenables en aval de l'insert. Cette Demande de Brevet men
tionne, en outre, que l'un des avantages majeur de l'invention est de pourvoir à des séquences d'ADN dérivés de l'ARN génomique du virus de la rage qui, contrairement au génome lui-même, peuvent être isolées sous une forme dépourvue de nucléoprotéines. Les ADNc selon cette Demande de Brevet peuvent être utilisés en tant que sondes, par exemple pour la détection de la présence dans un fluide biologique, du virus de la rage, par hybridation. This Application also covers recombinant DNAs which contain one of the abovementioned inserts placed under the control of a promoter derived from the genome of the SV40 virus, which are vectors which can be used to transform eukaryotic cells (such as Vero cells) and produce proteins endowed with immunological properties, the promoter being able, more generally, to be a viral or eukaryotic promoter recognized by the polymerases of the selected cells and which additionally comprises suitable polyadenylation sites downstream of the insert. This Patent Application men Furthermore, one of the major advantages of the invention is to provide DNA sequences derived from the genomic RNA of the rabies virus which, unlike the genome itself, can be isolated under a form devoid of nucleoproteins. The cDNAs according to this Patent Application can be used as probes, for example for the detection of the presence in a biological fluid, of the rabies virus, by hybridization.
Les polypeptides N, M1, M2 tels que définis dans cette Demande de Brevet et les Articles précités, par leurs structures peptidiques, peuvent être utilisés pour produire des anticorps correspondants qui peuvent être eux-mêmes utilisés pour le diagnostic in vitro de la présence de polypeptides viraux dans un fluide biologique, la protéine M1 présentant un intérêt tout particulier, notamment en tant que composition immunogène en association avec un véhicule utilisé dans la production de vaccins. The N, M1, M2 polypeptides as defined in this Patent Application and the aforementioned Articles, by their peptide structures, can be used to produce corresponding antibodies which can themselves be used for the in vitro diagnosis of the presence of polypeptides viral in a biological fluid, the protein M1 being of very particular interest, in particular as an immunogenic composition in association with a vehicle used in the production of vaccines.
N. TORDO et al., décrivent dans un Article paru dans Virol., 1988, 165, 565-576, la détermination de la séquence complète du génome du virus rabique et ont trouvé qu'il existe des domaines hautement conservés dans les protéines L (polymérase) des virus à ARN non segmente négatif. N. TORDO et al., In an article published in Virol., 1988, 165, 565-576, describe the determination of the complete genome sequence of the rabies virus and have found that there are highly conserved domains in the L proteins. (polymerase) of non-segmented negative RNA viruses.
L'Article au nom de O. POCH et al. paru dans Biochimie, 1988, 70, 1019-1029 décrit des fragments d'ADNc du génome d'une souche avirulente (AVO1) du virus rabique. La séquence de 3 386 nucléotides à partir de l'extrémité 3' couvre les gènes codant pour l'ARN leader, la nucléoprotéine N, la phosphoprotéine M1 et la protéine matrice M2, ainsi que les régions intergéniques. La comparaison de la séquence AVO1 avec celle d'autres souches du virus de la rage révèle une conservation importante aussi bien au niveau des nucléotides que des acides aminés.
La comparaison du génome rabique avec ceux d'autres virus à ARN monocaténaire négatif non segmentés (rhabdovirus et paramyxovirus) indique que les signaux d'initiation et d'arrêt de transcription, localisés aux extrémités de chaque gène codant pour une protéine, et les régions de la phosphoprotéine et des protéines matrices qui pourraient être impliquées dans le processus de transcription conservent une structure globale similaire. The article in the name of O. POCH et al. published in Biochemistry, 1988, 70, 1019-1029 describes cDNA fragments of the genome of an avirulent strain (AVO1) of the rabies virus. The 3,386 nucleotide sequence from the 3 'end covers the genes encoding the leader RNA, nucleoprotein N, phosphoprotein M1 and the matrix protein M2, as well as the intergenic regions. Comparison of the AVO1 sequence with that of other strains of the rabies virus reveals significant conservation both at the nucleotide and amino acid level. Comparison of the rabies genome with those of other non-segmented negative single-stranded RNA viruses (rhabdovirus and paramyxovirus) indicates that the signals for initiation and stop of transcription, located at the ends of each gene coding for a protein, and the regions phosphoprotein and matrix proteins which could be involved in the transcription process maintain a similar overall structure.
Les résultats énoncés dans cet Article suggèrent que les caractéristiques distinctives de la transcription du virus de la rage se trouvent dans les régions intergéniques nettement variables. The results reported in this article suggest that the distinctive features of rabies virus transcription are found in clearly variable intergenic regions.
Le virus Mokola est un virus rabique dit "apparenté" : les vaccins rabiques ne confèrent qu'une très faible protection à l'égard de l'infection par le virus Mokola. The Mokola virus is a so-called "related" rabies virus: rabies vaccines provide very little protection against infection by the Mokola virus.
Les échecs des vaccinations ont été reproduits au laboratoire : des souris vaccinées par les trois vaccins disponibles sur le marché (souche PV (Pasteur) ; souche PM (Mérieux) ; souche Flury LEP (Behring)) ne résistent pas à une épreuve par le virus Mokola. De fait, leur sérum ne contient que peu d'anticorps capables de neutraliser le virus Mokola in vitro, et leurs lymphocytes présentent une faible cytotoxicité vis-à-vis de cellules cibles infectées par le virus Mokola. Vaccination failures have been reproduced in the laboratory: mice vaccinated with the three vaccines available on the market (PV strain (Pasteur); PM strain (Mérieux); Flury LEP strain (Behring)) do not withstand a test by the virus Mokola. In fact, their serum contains only few antibodies capable of neutralizing the Mokola virus in vitro, and their lymphocytes exhibit low cytotoxicity with respect to target cells infected with the Mokola virus.
On peut citer notamment l'Article au nom de T.J. WIKTOR et al., paru dans Develop. Biol. Stand., 1984, 57, 199-211, qui décrit l'analyse antigénique du virus de la rage et du virus Mokola par l'utilisation d'anticorps monoclonaux et qui précise que contrairement à d'autres virus proches du virus rabique, le virus Mokola est peu neutralisé par le sérum de personnes vaccinées contre la rage. One can quote in particular the Article in the name of T.J. WIKTOR et al., Published in Develop. Biol. Stand., 1984, 57, 199-211, which describes the antigenic analysis of the rabies virus and of the Mokola virus by the use of monoclonal antibodies and which specifies that unlike other viruses close to the rabies virus, the Mokola virus is little neutralized by the serum of people vaccinated against rabies.
On peut citer également l'Article de E. CELIS et al., paru dans J. Virol., 1988, 3 128-3 134 qui pré
cise que les cellules mononucléées du sang périphérique humain, et les clones T d'individus immunisés par un vaccin antirabique PM ont été testées pour leur capacité à reconnaître les déterminants antigéniques des virus de la rage et des virus apparentés à la rage, dans un test de prolifération induit par l'antigène (AIPA). Quelques cellules T mais pas toutes présentent des réactions croisées avec différentes souches de laboratoire du virus rabique et avec des virus apparentés tels que Duvenhage et Mokola. Ces cellules T réagissent avec des epitopes soit de la ribonucléoprotéine, soit de la glycoprotéine virale. We can also cite the Article by E. CELIS et al., Published in J. Virol., 1988, 3 128-3 134 which pre states that human peripheral blood mononuclear cells, and T clones of individuals immunized with a PM rabies vaccine have been tested for their ability to recognize the antigenic determinants of rabies and rabies-related viruses in a test of proliferation induced by the antigen (AIPA). Some but not all T cells cross-react with different laboratory strains of the rabies virus and with related viruses such as Duvenhage and Mokola. These T cells react with epitopes of either ribonucleoprotein or viral glycoprotein.
Aucune de ces publications ne concerne la production de clones d'ADNc du génome d'un virus apparenté au virus rabique, tel que le virus Mokola, ni leur utilisation en tant qu'agent de prévention, de traitement ou de diagnostic. None of these publications relates to the production of cDNA clones of the genome of a virus related to the rabies virus, such as the Mokola virus, nor to their use as an agent for prevention, treatment or diagnosis.
De plus, certains de ces Articles montrent que, bien qu'il existe une réactivité croisée entre le virus rabique et des virus apparentés au virus rabique, le vaccin anti-rabique ne protège pas contre les virus apparentés tels que le virus Mokola. In addition, some of these Articles show that, although there is cross-reactivity between the rabies virus and viruses related to the rabies virus, the rabies vaccine does not protect against related viruses such as the Mokola virus.
Il apparaît donc nécessaire de proposer un vaccin contre ce virus à usage humain et/ou vétérinaire, tant pour prévenir l'infection des populations à risque It therefore appears necessary to offer a vaccine against this virus for human and / or veterinary use, both to prevent infection of populations at risk
(vétérinaires, éleveurs, travailleurs de laboratoire) et les animaux domestiques, que pour traiter les personnes après exposition au risque d'infection. (veterinarians, breeders, laboratory workers) and pets, only to treat people after exposure to the risk of infection.
Il ne semble pas qu'un vaccin inactivé puisse actuellement être réalisé à l'échelle industrielle, en raison du faible titre viral obtenu en culture cellulaire, de l'ordre de 107-108. It does not seem that an inactivated vaccine can currently be produced on an industrial scale, due to the low viral titer obtained in cell culture, of the order of 10 7 -10 8 .
C'est pourquoi la préparation d'un vaccin anti-Mokola par génie génétique présente un intérêt majeur.
En ce qui concerne le diagnostic de la rage, deux types de méthodes sont actuellement utilisés pour mettre en évidence la présence du virus rabique dans un prélèvement suspect : This is why the preparation of a Mokola vaccine by genetic engineering is of major interest. Regarding the diagnosis of rabies, two types of methods are currently used to detect the presence of rabies virus in a suspect sample:
- on peut citer notamment la méthode qui utilise des anticorps pour la recherche d'antigène rabique, soit par immunofluorescence (IF), soit par un test immunoenzymatique (RREID) ; et - Mention may in particular be made of the method which uses antibodies for the search for rabies antigen, either by immunofluorescence (IF), or by an immunoenzymatic test (RREID); and
- la mise en évidence du virus, soit par inoculation à la souris, soit par son isolement sur culture cellulaire (CC). - the detection of the virus, either by inoculation with the mouse, or by its isolation on cell culture (CC).
On dispose actuellement d'une batterie d'anticorps monoclonaux qui permettent d'identifier les différentes souches de ragé. Certains de ces anticorps monoclonaux sont spécifiques de certains serotypes au sein des Lyssavirus et même plus précisément d'isolats au sein d'un même serotype. Ces anticorps monoclonaux permettent donc une caractérisation précise des différentes souches de Lyssavirus qui est applicable dans les enquêtes épidémiologiques. Néanmoins l'identification des souches permise par cette technique est longue car elle nécessite généralement une adaptation préalable de ces souches à la culture cellulaire. D'autre part chaque nouvel isolât appelle la mise en oeuvre de fusions pour la production de nouveaux anticorps monoclonaux spécifiques. We currently have a battery of monoclonal antibodies which allow us to identify the different strains of rabies. Some of these monoclonal antibodies are specific for certain serotypes within Lyssaviruses and even more specifically for isolates within the same serotype. These monoclonal antibodies therefore allow a precise characterization of the different strains of Lyssavirus which is applicable in epidemiological surveys. However, the identification of strains allowed by this technique is long since it generally requires prior adaptation of these strains to cell culture. On the other hand, each new isolate calls for the implementation of fusions for the production of new specific monoclonal antibodies.
A. ERMINE et al. dans un Article paru dans Mol. Cell. Probes, 1988, 2, 75-82 décrivent une méthode de diagnostic rapide de l'infection rabique par une méthode d'hybridation. A. ERMINE et al. in an article published in Mol. Cell. Probes, 1988, 2, 75-82 describe a method of rapid diagnosis of rabies infection by a hybridization method.
Cette méthode d'hybridation est utilisée pour détecter les transcrits rabiques dans le cerveau. Des sondes d'ADNc marquées au 32p provenant du génome de la souche rabique PV (serotype 1) sont utilisées pour identifier des quantités très faibles d'ARN viral spécifique. L'ARN viral purifié est obtenu après extraction phénolique. L'ARN est fixé sur des membranes en nylon et
hybride avec un pool d'inserts M13 complémentaires à 200- 400 nucléotides de chaque gène rabique et de chaque ARNm. Les sondes marquées hybridées sont détectées par autoradiographie. Une hybridation a été observée avec des prélèvements de cerveau d'animaux inoculés par des souches vulpines de virus rabiques (serotype 1). Une réponse positive a été obtenue pour des quantités d'ARN total de 80 ng. La détection des transcrits viraux est restée possible sur des cerveaux prélevés une semaine après la mort de l'animal. Une corrélation totale est observée en comparaison avec d'autres techniques telles que la détection d'antigènes rabiques par l'utilisation d'un anticorps antirabique fluorescent ou l'isolement du virus sur cellules de neuroblastomè murin. This hybridization method is used to detect rabid transcripts in the brain. 32 p-labeled cDNA probes from the genome of the rabies strain PV (serotype 1) are used to identify very small amounts of specific viral RNA. The purified viral RNA is obtained after phenolic extraction. The RNA is fixed on nylon membranes and hybrid with a pool of M13 inserts complementary to 200-400 nucleotides of each rabies gene and each mRNA. The labeled hybridized probes are detected by autoradiography. Hybridization was observed with brain samples from animals inoculated with vulpine strains of rabies virus (serotype 1). A positive response was obtained for amounts of total RNA of 80 ng. Detection of viral transcripts remained possible on brains taken a week after the animal's death. A total correlation is observed in comparison with other techniques such as the detection of rabies antigens by the use of a fluorescent anti-rabies antibody or the isolation of the virus on murine neuroblastome cells.
Cependant ces méthodes ne permettent pas d'une part, la détection rapide d'une infection à Lyssavirus à partir d'un prélèvement de salive ou d'organe et en particulier, la détection et l'identification du virus Mokola, directement dans un échantillon biologique. However, these methods do not allow on the one hand, the rapid detection of a Lyssavirus infection from a saliva or organ sample and in particular, the detection and identification of the Mokola virus, directly in a sample organic.
Lors de la 7ème Rencontre Internationale sur les virus monocaténaires négatifs, qui a eu lieu à Dinard, FRANCE, du 18 au 23 septembre 1988, les Inventeurs ont présenté une communication dans laquelle ils ont fait état de leurs travaux de clonage du génome du virus Mokola, dans le but de pouvoir produire un vaccin par génie génétique. L'abstract de cette Communication indique que des virus Mokola recueillis dans le surnageant de cellules BHK21 infectées, ont été purifiés et l'ARN génomique en a été extrait. During the 7th International Meeting on negative single-stranded viruses, which took place in Dinard, FRANCE, from September 18 to 23, 1988, the Inventors presented a communication in which they reported on their work on cloning the genome of the Mokola virus. , in order to be able to produce a vaccine by genetic engineering. The abstract of this Communication indicates that Mokola viruses collected in the supernatant of infected BHK21 cells have been purified and the genomic RNA has been extracted therefrom.
La présente invention s'est donné pour but de pourvoir à un vaccin spécifique contre le virus Mokola obtenu en exprimant des antigènes viraux majoritairement impliqués dans la réponse immunitaire ainsi qu'un vaccin polyvalent dirigé contre tous les serotypes de Lyssavirus, l'obtention par génie génétique ayant pour avantage de résoudre les problèmes de production de virus et de
fournir un agent de diagnostic spécifique et très sensible à l'égard des infections à Lyssavirus. The purpose of the present invention is to provide a specific vaccine against the Mokola virus obtained by expressing viral antigens mainly involved in the immune response as well as a polyvalent vaccine directed against all the Lyssavirus serotypes, obtaining by engineering The advantage of genetics is that it solves the problems of virus production and provide a specific diagnostic agent that is very sensitive to Lyssavirus infections.
C'est également un but de l'invention de pourvoir à un procédé de détection rapide d'une infection à Lyssavirus à partir d'un prélèvement de salive ou d'organe. It is also an object of the invention to provide a method for rapid detection of a Lyssavirus infection from a saliva or organ sample.
La présente invention a pour objet un procédé de détection et/ou d'identification rapide de faibles quantités de Lyssavirus présents dans un échantillon biologique, caractérisé en ce que ledit échantillon convenablement traité pour extraire l'ARN viral et/ou les produits de transcription des Lyssavirus éventuellement présents est : The subject of the present invention is a process for the rapid detection and / or identification of small amounts of Lyssavirus present in a biological sample, characterized in that said sample suitably treated to extract the viral RNA and / or the transcription products of the Lyssavirus possibly present is:
(1) mis en contact avec au moins une amorce appropriée des Lyssavirus, pour obtenir l'ADNc de l'ARN génomique ou de l'ARNm ; (1) brought into contact with at least one appropriate primer of Lyssaviruses, to obtain the cDNA of the genomic RNA or of the mRNA;
(2) puis la séquence d'ADNc est mise en contact avec une paire d'amorces appropriées des Lyssavirus pour amplifier au moins un fragment dudit ADNc, l'une desdites amorces étant différente de celle de l'étape (1) et l'autre amorce étant identique à ou différente de celle de l'étape (1) ; (2) then the cDNA sequence is brought into contact with a pair of suitable Lyssavirus primers to amplify at least one fragment of said cDNA, one of said primers being different from that of step (1) and the other primer being identical to or different from that of step (1);
(3) après quoi, la séquence d'ADNc amplifiée est détectée par un moyen approprié. (3) after which, the amplified cDNA sequence is detected by appropriate means.
La méthode PCR est notamment décrite par SAIKI et al. dans Science, 1988,239, 487 ainsi que dans les Demandes de Brevet Européen CETUS Nº 200 362 et 201 184. Cependant le procédé de la présente invention permet de manière inattendue de détecter rapidement (moins de 12 heures) une infection à Lyssavirus à partir d'un prélèvement de salive ou d'organe et par la suite d'identifier le Lyssavirus. The PCR method is notably described by SAIKI et al. in Science, 1988, 239, 487 as well as in European Patent Applications CETUS Nº 200 362 and 201 184. However, the method of the present invention unexpectedly allows rapid detection (less than 12 hours) of a Lyssavirus infection from a saliva or organ sample and then identify the Lyssavirus.
Selon un mode de mise en oeuvre avantageux dudit procédé de détection, les amorces sont choisies dans le groupe qui comprend les amorces spécifiques d'une souche, pour un serotype de Lyssavirus, les amorces spé
cifiques d'un serotype de Lyssavirus et les amorces constituées d'une séquence conservée d'un gène commune à tous les Lyssavirus et ci-après dénommées amorces polyvalentes. According to an advantageous embodiment of said detection method, the primers are chosen from the group which comprises the primers specific for a strain, for a serotype of Lyssavirus, the specific primers specific for a Lyssavirus serotype and the primers consisting of a conserved sequence of a gene common to all Lyssaviruses and hereinafter called polyvalent primers.
Parmi les amorces polyvalentes elles-mêmes, on peut distinguer au sens de la présente invention, les amorces de diagnostic ou de détection d'un Lyssavirus (infection à Lyssavirus : réponse du type Oui/Non) et les amorces de diagnostic différentiel ou typage (infection à Lyssavirus : précision sur le type). Among the versatile primers themselves, one can distinguish, within the meaning of the present invention, the primers for diagnosis or detection of a Lyssavirus (Lyssavirus infection: response of the Yes / No type) and the primers for differential diagnosis or typing ( Lyssavirus infection: details on type).
Selon une disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce rabique 3', localisée en position 1-18 des génomes rabiques et Mokola. According to an advantageous arrangement of this embodiment, the primer is a 3 'rabies primer, located in position 1-18 of the rabies and Mokola genomes.
Selon une autre disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce rabique M2, localisée en position 2901-2918 du génome rabique. According to another advantageous arrangement of this embodiment, the primer is a rabies primer M2, located in position 2901-2918 of the rabies genome.
Selon une autre disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce rabique constituée de 23 nucléotides, localisée en position 4665- 4687 du génome rabique et en position 4675-4697 du génome Mokola, ci-après dénommée amorce G. According to another advantageous arrangement of this embodiment, the primer is a rabies primer consisting of 23 nucleotides, located in position 4665-4687 of the rabies genome and in position 4675-4697 of the Mokola genome, hereinafter called primer G .
Selon une autre disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce rabique constituée de 24 nucléotides, localisée en position 5520- 5543 du génome rabique et en position 5545-5568 du génome Mokola, ci-après dénommée amorce L. According to another advantageous arrangement of this embodiment, the primer is a rabies primer consisting of 24 nucleotides, located in position 5520-5543 of the rabies genome and in position 5545-5568 of the Mokola genome, hereinafter called primer L .
Selon encore une autre disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce Mokola ou rabique constituée de 18 nucléotides localisée en position 587-605 des génomes rabiques et Mokola, ciaprès dénommée amorce Na. According to yet another advantageous arrangement of this embodiment, the primer is a Mokola or rabic primer consisting of 18 nucleotides located in position 587-605 of the rabies and Mokola genomes, hereinafter called the Na primer.
Selon encore une autre disposition avantageuse de ce mode de mise en oeuvre, l'amorce est une amorce Mokola ou rabique constituée de 16 nucléotides localisée
en position 1013-1029 des génomes rabiques et Mokola, ciaprès dénommée amorce Nb. According to yet another advantageous arrangement of this embodiment, the primer is a Mokola or rabic primer consisting of 16 localized nucleotides in position 1013-1029 of the rabies and Mokola genomes, below called primer Nb.
Selon un mode de mise en oeuvre avantageux dudit procédé, la détection de la séquence nucléotidique amplifiée est réalisée par hybridation au moyen d'une sonde ou d'une batterie de sondes appropriées aux différents Lyssavirus . According to an advantageous embodiment of said method, the detection of the amplified nucleotide sequence is carried out by hybridization using a probe or a battery of probes suitable for the various Lyssaviruses.
Selon un autre mode de mise en oeuvre avantageux dudit procédé, la détection de la séquence nucléotidique amplifiée est réalisée par clivage de ladite séquence avec au moins une batterie d'enzymes de restriction appropriées suivie de la séparation par électrophorèse des fragments obtenus. According to another advantageous embodiment of said method, the detection of the amplified nucleotide sequence is carried out by cleavage of said sequence with at least one battery of suitable restriction enzymes followed by separation by electrophoresis of the fragments obtained.
Selon une disposition avantageuse de ce mode de mise en oeuvre, la batterie d'enzymes comprend BamH I, Hind II, Hind III, Pst I. According to an advantageous arrangement of this embodiment, the battery of enzymes comprises BamH I, Hind II, Hind III, Pst I.
Selon une autre disposition avantageuse de ce mode de mise en oeuvre, la batterie d'enzymes comprend Rsa I, Taq I, BstX I, Fnu4H I. According to another advantageous arrangement of this embodiment, the battery of enzymes comprises Rsa I, Taq I, BstX I, Fnu4H I.
L'analyse des homologies de séquence entre les serotypes 1 et 3, qui sont actuellement les plus divergents parmi les Lyssavirus, permet de définir des zones conservées ou variables, orientant le choix respectivement vers les amorces spécifiques ou polyvalentes. Analysis of the sequence homologies between serotypes 1 and 3, which are currently the most divergent among Lyssaviruses, makes it possible to define conserved or variable zones, orienting the choice respectively towards specific or polyvalent primers.
Dans le cas où les amorces polyvalentes choisies permettent l'amplification d'une zone conservée d'un Lyssavirus, le procédé de détection rapide, conforme à l'invention, de faibles quantités de Lyssavirus dans un échantillon , a l'avantage de permettre de porter un diagnostic d'infection à Lyssavirus sur des infections débutantes ou sur des prélèvements de salive effectués chez les animaux domestiques suspects d'être responsables d'une contamination humaine, plusieurs jours avant leur mort, contrairement à la pratique actuelle ; en effet, par exemple, la paire d'amorces, amorce Na et amorce Nb telles que définies dans l'invention, situées sur le gène
N et peu distantes l'une de l'autre (400 pb) permet la détection (réponse Oui/Non) d'un Lyssavirus même présent en très petites quantités et ce, même si l'échantillon est très dégradé. En effet, la sensibilité des techniques habituelles est relativement faible par rapport à la méthode conforme à l'invention, qui est rapide et permet de porter un diagnostic en moins de 12 heures. In the case where the polyvalent primers chosen allow the amplification of a conserved area of a Lyssavirus, the rapid detection method, according to the invention, of small amounts of Lyssavirus in a sample, has the advantage of making it possible to diagnose Lyssavirus infection on early infections or on saliva samples taken from domestic animals suspected of being responsible for human contamination, several days before their death, contrary to current practice; indeed, for example, the pair of primers, Na primer and Nb primer as defined in the invention, located on the gene N and not far apart from one another (400 bp) allows the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
Dans le cas où les amorces choisies permettent la synthèse et l'amplification d'une zone variable (par exemple, pseudogène psi ou certaines régions du gène M1) d'un Lyssavirus, le procédé conforme à l'invention permet une identification et un typage du Lyssavirus impliqué dans l'infection à détecter. En effet, la paire d'amorces, amorce rabique G et amorce rabique L telles que définies ci-dessus, associée à une batterie d'enzymes de restriction appropriées, permet de typer aussi bien les différentes souches de virus de la rage, le virus Mokola, que les souches de Lyssavirus isolées de chauvesouris ; les deux amorces précitées sont choisies de manière à correspondre à des zones conservées chez les Lyssavirus, l'une dans la partie distale du gène G, l'autre dans la partie proximale du gène L, pour permettre l'amplification de la région qui les sépare, d'environ 860 nucléotides (pseudogène psi), sur toutes les souches rabiques ainsi que sur Mokola. La bande amplifiée est détectée soit par hybridation avec une sonde plus ou moins spécifique de souche appropriée, soit par hydrolyse en présence d'enzymes de restriction plus ou moins spécifiques de souche. In the case where the primers chosen allow the synthesis and the amplification of a variable zone (for example, pseudogenic psi or certain regions of the M1 gene) of a Lyssavirus, the method according to the invention allows identification and typing of the Lyssavirus involved in the infection to be detected. Indeed, the pair of primers, rabies primer G and rabies primer L as defined above, associated with a battery of appropriate restriction enzymes, makes it possible to type both the different strains of rabies virus, the virus Mokola, as strains of Lyssavirus isolated from bats; the two aforementioned primers are chosen so as to correspond to conserved zones in Lyssaviruses, one in the distal part of the G gene, the other in the proximal part of the L gene, to allow the amplification of the region which separates, around 860 nucleotides (pseudogen psi), on all rabies strains as well as on Mokola. The amplified band is detected either by hybridization with a more or less specific strain-specific probe, or by hydrolysis in the presence of more or less strain-specific restriction enzymes.
On peut en outre appliquer directement sur le morceau d'agarose contenant la bande amplifiée, une technique de séquençage directe. In addition, a direct sequencing technique can be applied directly to the piece of agarose containing the amplified band.
La présente invention a également pour objet les séquences spécifiques du virus Mokola, lesquelles séquences ont permis de mettre en évidence les zones conservées et les zones variables dans le genre Lyssa
virus et par comparaison entre les génomes rabiques et Mokola, d'apprécier la variabilité de chaque région genomique et de mettre au point le procédé de détection et/ou d'identification de Lyssavirus ci-dessus. La séquence nucleotidique de l'ADNc de l'ARN genomique du virus Mokola, est caractérisée en ce qu'elle comprend environ 12 000 nucléotides. The present invention also relates to the specific sequences of the Mokola virus, which sequences have made it possible to highlight the conserved areas and the variable areas in the genus Lyssa. virus and by comparison between the rabies and Mokola genomes, to assess the variability of each genomic region and to develop the detection and / or identification method of Lyssavirus above. The nucleotide sequence of the cDNA of the genomic RNA of the Mokola virus is characterized in that it comprises approximately 12,000 nucleotides.
Ladite séquence d'ADNc de l'ARN genomique est, en outre, caractérisée en ce que les extrémités 3' et 5' sont complémentaires, en ce que les 12 nucléotides de l'extrémité 5' sont identiques à ceux de la souche PV du virus rabique, et en ce qu'elle présente successivement de 3' en 5' le gène codant pour l'ARN "leader" puis les gènes codant pour les protéines N, M1, M2, G et L.
Said genomic RNA cDNA sequence is further characterized in that the 3 'and 5' ends are complementary, in that the 12 nucleotides of the 5 'end are identical to those of the PV strain of rabies virus, and in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the proteins N, M1, M2, G and L.
Selon un mode de réalisation avantageux de ladite séquence de l'ADNc de l'ARN genomique du virus Mokola, celle-ci comprend la séquence en nucléotides et la séquence déduite en amino-acides suivante (I) : According to an advantageous embodiment of said sequence of the cDNA of the genomic RNA of the Mokola virus, it comprises the sequence in nucleotides and the deduced sequence in amino acids as follows (I):
leader ARN RNA leader
ACGCTTAACA ACCAGATCAA AGAAGACACA GATAGTATCA GTGACCTAAA 50 ACGCTTAACA ACCAGATCAA AGAAGACACA GATAGTATCA GTGACCTAAA 50
> Met-Glu-Ser-Asp-Lys-Ile-Val-Phe > Met-Glu-Ser-Asp-Lys-Ile-Val-Phe
CAAAATGT
ATG GAG TCT GAC AAG ATT GTG TTC 94 Lys-Val-Asn-Asn-Gln-Val-Val-Ser-Leu-Lys-Pro-Glu-Val-Ile-CAAAATGT ATG GAG TCT GAC AAG ATT GTG TTC 94 Lys-Val-Asn-Asn-Gln-Val-Val-Ser-Leu-Lys-Pro-Glu-Val-Ile-
AAG GTG AAT AAC CAA GTT GTT TCT TTG AAG CCT GAG GTC ATA 136AAG GTG AAT AAC CAA GTT GTT TCT TTG AAG CCT GAG GTC ATA 136
Ser-Asp-Gln-Tyr-Glu-Tyr-Lys-Tyr-Pro-Ala-Ile-Leu-Asp-Gly Ser-Asp-Gln-Tyr-Glu-Tyr-Lys-Tyr-Pro-Ala-Ile-Leu-Asp-Gly
TCA GAT CAA TAT GAG TAT AAA TAT CCC GCC ATT CTA GAT GGG 178 TCA GAT CAA TAT GAG TAT AAA TAT CCC GCC ATT CTA GAT GGG 178
Lys-Lys-Pro-Gly-Ile-Thr-Leu-Gly.-Lys-Ala-Pro-Asp-Leu-Asn-Lys-Lys-Pro-Gly-Ile-Thr-Leu-Gly.-Lys-Ala-Pro-Asp-Leu-Asn-
AAG AAA CCA GGG ATC ACC TTG GGG AAG GCA CCT GAT CTA AAC 220AAG AAA CCA GGG ATC ACC TTG GGG AAG GCA CCT GAT CTA AAC 220
Thr-Ala-Tyr-Lys-Ser-Ile-Leu-Ser-Gly-Met-Lys-Ala-Ala-Lys-Thr-Ala-Tyr-Lys-Ser-Ile-Leu-Ser-Gly-Met-Lys-Ala-Ala-Lys-
ACT GCA TAC AAA TCC ATC CTA TCA GGT ATG AAG GCT GCA AAG 262ACT GCA TAC AAA TCC ATC CTA TCA GGT ATG AAG GCT GCA AAG 262
Leu-Asp-Pro-Asp-Asp-Val-Cys-Ser-Tyr-Leu-Ala-Ala-Ala-Met- CTT GAC CCA GAC GAT GTT TGC TCT TAC TTA GCA GCT GCT ATG 304Leu-Asp-Pro-Asp-Asp-Val-Cys-Ser-Tyr-Leu-Ala-Ala-Ala-Met- CTT GAC CCA GAC GAT GTT TGC TCT TAC TTA GCA GCT GCT ATG 304
His-Leu-Phe-Glu-Gly-Val-Cys-Pro-Glu-Asp-Trp-Val-Ser-Tyr- CAT CTA TTC GAG GGG GTC TGT CCC GAG GAC TGG GTT AGT TAT 346His-Leu-Phe-Glu-Gly-Val-Cys-Pro-Glu-Asp-Trp-Val-Ser-Tyr- CAT CTA TTC GAG GGG GTC TGT CCC GAG GAC TGG GTT AGT TAT 346
Gly-Ile-Val-Ile-Ala-Lys-Lys-Gly-Glu-Lys-Ile-Asn-Pro-Ser- GGG ATT GTC ATT GCG AAG AAG GGA GAG AAA ATC AAC CCC AGC 388Gly-Ile-Val-Ile-Ala-Lys-Lys-Gly-Glu-Lys-Ile-Asn-Pro-Ser- GGG ATT GTC ATT GCG AAG AAG GGA GAG AAA ATC AAC CCC AGC 388
Val-Ile-Val-Asp-Ile-Val-Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp- GTG ATC GTC GAT ATA GTT CGC ACT AAC GTT GAG GGG AAT TGG 430Val-Ile-Val-Asp-Ile-Val-Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp- GTG ATC GTC GAT ATA GTT CGC ACT AAC GTT GAG GGG AAT TGG 430
Ala-Gln-Ala-Gly-Gly-Thr-Asp-Val-Ile-Arg-Asp-Pro-Thr-Met- GCT CAA GCG GGA GGA ACT GAT GTG ATT AGA GAT CCT ACA ATG 472Ala-Gln-Ala-Gly-Gly-Thr-Asp-Val-Ile-Arg-Asp-Pro-Thr-Met- GCT CAA GCG GGA GGA ACT GAT GTG ATT AGA GAT CCT ACA ATG 472
Ala-Glu-His-Ala-Ser-Leu-Val-Gly-Leu-Leu-Leu-Cys-Leu-Tyr- GCA GAG CAT GCT TCA TTG GTC GGA CTG TTA TTA TGT CTG TAT 514Ala-Glu-His-Ala-Ser-Leu-Val-Gly-Leu-Leu-Leu-Cys-Leu-Tyr- GCA GAG CAT GCT TCA TTG GTC GGA CTG TTA TTA TGT CTG TAT 514
Arg-Leu-Ser-Lys-Ile-Val-Gly-Gln-Asn-Thr-Ala-Asn-Tyr-Lys-Arg-Leu-Ser-Lys-Ile-Val-Gly-Gln-Asn-Thr-Ala-Asn-Tyr-Lys-
CGA TTG AGC AAG ATA GTC GGT CAG AAC ACA GCA AAC TAT AAA 556CGA TTG AGC AAG ATA GTC GGT CAG AAC ACA GCA AAC TAT AAA 556
Thr-Asn-Val-Ala-Asp-Arg-Met-Glu-Gln-Ile-Phe-Glu-Thr-Ala-Thr-Asn-Val-Ala-Asp-Arg-Met-Glu-Gln-Ile-Phe-Glu-Thr-Ala-
ACC AAT GTA GCA GAC AGA ATG GAA CAA ATA TTT GAG ACT GCT 598ACC AAT GTA GCA GAC AGA ATG GAA CAA ATA TTT GAG ACT GCT 598
Pro-Phe-Ala-Lys-Val-Val-Glu-His-His-Thr-Leu-Met-Thr-Thr-Pro-Phe-Ala-Lys-Val-Val-Glu-His-His-Thr-Leu-Met-Thr-Thr-
CCT TTT GCG AAG GTG GTG GAA CAT CAC ACA TTG ATG ACT ACT 640CCT TTT GCG AAG GTG GTG GAA CAT CAC ACA TTG ATG ACT ACT 640
His-Lys-Met-Cys-Ala-Asn-Trp-Ser-Thr-Ile-Pro-Asn-Phe-Arg-His-Lys-Met-Cys-Ala-Asn-Trp-Ser-Thr-Ile-Pro-Asn-Phe-Arg-
CAT AAG ATG TGC GCT AAC TGG AGC ACT ATA CCT AAC TTC AGA 682
Phe-Leu-Val-Gly-Thr-Tyr-Asp-Met-Phe-Phe-Ala-Arg-Val-Glu- TTC CTG GTG GGC ACA TAT GAT ATG TTC TTT GCA AGA GTC GAG 724CAT AAG ATG TGC GCT AAC TGG AGC ACT ATA CCT AAC TTC AGA 682 Phe-Leu-Val-Gly-Thr-Tyr-Asp-Met-Phe-Phe-Ala-Arg-Val-Glu- TTC CTG GTG GGC ACA TAT GAT ATG TTC TTT GCA AGA GTC GAG 724
His-Ile-Tyr-Ser-Ala-Leu-Arg-Val-Gly-Thr-Val-Val-Thr-Ala-His-Ile-Tyr-Ser-Ala-Leu-Arg-Val-Gly-Thr-Val-Val-Thr-Ala-
CAT ATA TAT TCG GCT CTC AGA GTC GGA ACA GTC GTG ACA GCC 766CAT ATA TAT TCG GCT CTC AGA GTC GGA ACA GTC GTG ACA GCC 766
Tyr-Glu-Asp-Cys-Ser-Gly-Leu-Val-Ser-Phe-Thr-Gly-Phe-Ile-Tyr-Glu-Asp-Cys-Ser-Gly-Leu-Val-Ser-Phe-Thr-Gly-Phe-Ile-
TAC GAG GAT TGC TCA GGC TTG GTC TCC TTT ACC GGG TTT ATC 808TAC GAG GAT TGC TCA GGC TTG GTC TCC TTT ACC GGG TTT ATC 808
Lys-Gln-Ile-Asn-Leu-Ser-Pro-Arg-Asp-Ala-Leu-Leu-Tyr-Phe-Lys-Gln-Ile-Asn-Leu-Ser-Pro-Arg-Asp-Ala-Leu-Leu-Tyr-Phe-
AAA CAA ATC AAT CTA TCT CCT AGA GAT GCA CTG CTA TAT TTC 850AAA CAA ATC AAT CTA TCT CCT AGA GAT GCA CTG CTA TAT TTC 850
Phe-His-Lys-Asn-Phe-Glu-Gly-Glu-Ile-Lys-Arg-Met-Phe-Glu-Phe-His-Lys-Asn-Phe-Glu-Gly-Glu-Ile-Lys-Arg-Met-Phe-Glu-
TTC CAT AAA AAC TTT GAA GGG GAG ATT AAG AGA ATG TTT GAG 892TTC CAT AAA AAC TTT GAA GGG GAG ATT AAG AGA ATG TTT GAG 892
Pro-Gly-Gln-Glu-Thr-Ala-Val-Pro-His-Ser-Tyr-Phe-Ile-His- CCG GGG CAA GAA ACA GCA GTT CCC CAC TCA TAC TTC ATT CAT 934Pro-Gly-Gln-Glu-Thr-Ala-Val-Pro-His-Ser-Tyr-Phe-Ile-His- CCG GGG CAA GAA ACA GCA GTT CCC CAC TCA TAC TTC ATT CAT 934
Phe-Arg-Ala-Leu-Gly-Leu-Ser-Gly-Lys-Ser-Pro-Tyr-Ser-Ser-Phe-Arg-Ala-Leu-Gly-Leu-Ser-Gly-Lys-Ser-Pro-Tyr-Ser-Ser-
TTT AGA GCA CTT GGC CTG AGT GGC AAG TCC CCG TAC TCG TCC 976 Asn-Ala-Val-Gly-His-Thr-Phe-Asn-Leu-Ile-His-Phe-Val-Gly-TTT AGA GCA CTT GGC CTG AGT GGC AAG TCC CCG TAC TCG TCC 976 Asn-Ala-Val-Gly-His-Thr-Phe-Asn-Leu-Ile-His-Phe-Val-Gly-
AAT GCT GTA GGT CAT ACT TTC AAT TTA ATC CAC TTT GTA GGA 1018AAT GCT GTA GGT CAT ACT TTC AAT TTA ATC CAC TTT GTA GGA 1018
Cys-Tyr-Met-Gly-Gln-Ile-Arg-Ser-Leu-Asn-Ala-Thr-Val-Ile-Cys-Tyr-Met-Gly-Gln-Ile-Arg-Ser-Leu-Asn-Ala-Thr-Val-Ile-
TGC TAT ATG GGT CAG ATC AGG TCT CTA AAT GCA ACT GTG ATC 1060TGC TAT ATG GGT CAG ATC AGG TCT CTA AAT GCA ACT GTG ATC 1060
Gln-Thr-Cys-Ala-Pro-Leu-Lys-Gly-Ala-Phe-Ser-Gln-Arg-Tyr-Gln-Thr-Cys-Ala-Pro-Leu-Lys-Gly-Ala-Phe-Ser-Gln-Arg-Tyr-
CAA ACA TGT GCA CCT CTC AAA GGT GCC TTT TCC CAA AGA TAT 1102CAA ACA TGT GCA CCT CTC AAA GGT GCC TTT TCC CAA AGA TAT 1102
Leu-Gly-Glu-Glu-Phe-Phe-Gly-Lys-Gly-Thr-Phe-Glu-Arg-Arg-Leu-Gly-Glu-Glu-Phe-Phe-Gly-Lys-Gly-Thr-Phe-Glu-Arg-Arg-
CTT GGA GAA GAG TTC TTT GGG AAA GGC ACC TTT GAG AGG AGG 1144CTT GGA GAA GAG TTC TTT GGG AAA GGC ACC TTT GAG AGG AGG 1144
Phe-Phe-Arg-Asp-Glu-Lys-Glu-Met-Gln-Asp-Tyr-Thr-Glu-Leu-Phe-Phe-Arg-Asp-Glu-Lys-Glu-Met-Gln-Asp-Tyr-Thr-Glu-Leu-
TTC TTT AGG GAT GAA AAA GAG ATG CAA GAT TAT ACA GAG CTT 1186 Glu-Glu-Ala-Arg-Val-Glu-Ala-Ser-Leu-Ala-Asp-Asp-Gly-Thr-TTC TTT AGG GAT GAA AAA GAG ATG CAA GAT TAT ACA GAG CTT 1186 Glu-Glu-Ala-Arg-Val-Glu-Ala-Ser-Leu-Ala-Asp-Asp-Gly-Thr-
GAG GAG GCC AGA GTA GAG GCT TCG CTC GCT GAT GAC GGG ACT 1228GAG GAG GCC AGA GTA GAG GCT TCG CTC GCT GAT GAC GGG ACT 1228
Val-Asp-Ser-Asp-Glu-Glu-Asp-Phe-Phe-Ser-Gly-Glu-Thr-Arg-Val-Asp-Ser-Asp-Glu-Glu-Asp-Phe-Phe-Ser-Gly-Glu-Thr-Arg-
GTA GAC TCA GAT GAG GAG GAC TTC TTC TCT GGA GAA ACC AGA 1270GTA GAC TCA GAT GAG GAG GAC TTC TTC TCT GGA GAA ACC AGA 1270
Ser-Pro-Glu-Ala-Val-Tyr-Ser-Arg-Ile-Met-Met-Asn-Asn-Gly-Ser-Pro-Glu-Ala-Val-Tyr-Ser-Arg-Ile-Met-Met-Asn-Asn-Gly-
AGT CCT GAA GCA GTT TAC AGT AGG ATA ATG ATG AAC AAC GGT 1312AGT CCT GAA GCA GTT TAC AGT AGG ATA ATG ATG AAC AAC GGT 1312
Lys-Leu-Lys-Lys-Val-His-Ile-Arg-Arg-Tyr-Ile-Ala-Val-Ser-Lys-Leu-Lys-Lys-Val-His-Ile-Arg-Arg-Tyr-Ile-Ala-Val-Ser-
AAA TTG AAG AAA GTT CAC ATA CGT AGG TAT ATT GCG GTG AGT 1354AAA TTG AAG AAA GTT CAC ATA CGT AGG TAT ATT GCG GTG AGT 1354
Ser-Asn-His-Gln-Ala-Arg-Pro-Asn-Ser-Phe-Ala-Glu-Phe-Leu-Ser-Asn-His-Gln-Ala-Arg-Pro-Asn-Ser-Phe-Ala-Glu-Phe-Leu-
TCT AAT CAT CAA GCG AGG CCG AAC TCT TTT GCA GAA TTC TTA 1396 Asn-Lys-Val-Tyr-Ala-Asp-Gly-Ser TCT AAT CAT CAA GCG AGG CCG AAC TCT TTT GCA GAA TTC TTA 1396 Asn-Lys-Val-Tyr-Ala-Asp-Gly-Ser
AAC AAG GTG TAT GCA GAT GGA TCA TAATCAGAGA GCTTCTTGGA 1440
AGACGATGAT CTATAGAGGG GTATTATTGT GAGACAGATT CC
1490 AAC AAG GTG TAT GCA GAT GGA TCA TAATCAGAGA GCTTCTTGGA 1440 AGACGATGAT CTATAGAGGG GTATTATTGT GAGACAGATT CC 1490
Ml
Met-Ser-Lys-Asp-Leu-Miss Met-Ser-Lys-Asp-Leu-
A]T
CCTCGAT TCGTGGTGTC AA ATG AGC AAA GAT TTG 1537A] T CCTCGAT TCGTGGTGTC AA ATG AGC AAA GAT TTG 1537
Val-His-Pro-Ser-Leu-Ile-Arg-Ala-Gly-Ile-Val-Glu-Leu-Glu-Val-His-Pro-Ser-Leu-Ile-Arg-Ala-Gly-Ile-Val-Glu-Leu-Glu-
GTG CAT CCT AGT CTT ATC AGG GCA GGG ATA GTA GAA CTG GAA 1580GTG CAT CCT AGT CTT ATC AGG GCA GGG ATA GTA GAA CTG GAA 1580
Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile-Asn-Arg-Thr-Ile-Glu-Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile-Asn-Arg-Thr-Ile-Glu-
ATG GCA GAA GAG ACT ACT GAT CTG ATT AAC AGG ACC ATA GAG 1622ATG GCA GAA GAG ACT ACT GAT CTG ATT AAC AGG ACC ATA GAG 1622
Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu-Pro-Leu-Tyr-Val-Asp-Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu-Pro-Leu-Tyr-Val-Asp-
AGC AAC CAA GCT CAC CTT CAG GGG GAG CCG CTT TAT GTT GAT 1664AGC AAC CAA GCT CAC CTT CAG GGG GAG CCG CTT TAT GTT GAT 1664
Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu-Arg-Ile-Glu-Asp-Lys-Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu-Arg-Ile-Glu-Asp-Lys-
TCA TTG CCG GAA GAT ATG AGC AGA TTG AGA ATA GAG GAC AAA 1706TCA TTG CCG GAA GAT ATG AGC AGA TTG AGA ATA GAG GAC AAA 1706
Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu-Glu-Arg-Asp-Glu-Gly- TCT CGT AGG ACT AAA ACA GAA GAA GAA GAA AGA GAT GAA GGT 1748Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu-Glu-Arg-Asp-Glu-Gly- TCT CGT AGG ACT AAA ACA GAA GAA GAA GAA AGA GAT GAA GGT 1748
Ser-Ser-Glu-Glu-Asp-Asn-Tyr-Leu-Ser-Glu-Gly-Gln-Asp-Pro- AGT TCT GAG GAG GAT AAC TAT TTG TCT GAG GGA CAA GAT CCA 1790Ser-Ser-Glu-Glu-Asp-Asn-Tyr-Leu-Ser-Glu-Gly-Gln-Asp-Pro- AGT TCT GAG GAG GAT AAC TAT TTG TCT GAG GGA CAA GAT CCA 1790
Leu-Ile-Pro-Phe-Gln-Asn-Phe-Leu-Asp-Glu-Ile-Gly-Ala-Arg-Leu-Ile-Pro-Phe-Gln-Asn-Phe-Leu-Asp-Glu-Ile-Gly-Ala-Arg-
TTA ATC CCC TTT CAG AAT TTC CTT GAT GAA ATT GGG GCC AGA 1832TTA ATC CCC TTT CAG AAT TTC CTT GAT GAA ATT GGG GCC AGA 1832
Ala-Val-Lys-Arg-Leu-Lys-Thr-Gly-Glu-Gly-Phe-Phe-Arg-Val- GCG GTC AAG AGA TTG AAG ACT GGC GAG GGA TTC TTC AGG GTG 1874Ala-Val-Lys-Arg-Leu-Lys-Thr-Gly-Glu-Gly-Phe-Phe-Arg-Val- GCG GTC AAG AGA TTG AAG ACT GGC GAG GGA TTC TTC AGG GTG 1874
Trp-Ser-Ala-Leu-Ser-Asp-Asp-Ile-Lys-Gly-Tyr-Val-Ser-Thr-Trp-Ser-Ala-Leu-Ser-Asp-Asp-Ile-Lys-Gly-Tyr-Val-Ser-Thr-
TGG TCT GCT CTG TCA GAT GAC ATA AAG GGG TAT GTA TCT ACC 1916TGG TCT GCT CTG TCA GAT GAC ATA AAG GGG TAT GTA TCT ACC 1916
Asn-Ile-Met-Thr-Ser-Gly-Glu-Arg-Asp-Thr-Lys-Ser-Ile-Gln-Asn-Ile-Met-Thr-Ser-Gly-Glu-Arg-Asp-Thr-Lys-Ser-Ile-Gln-
AAT ATA ATG ACA TCT GGG GAG AGA GAT ACT AAG AGC ATA CAA 1958AAT ATA ATG ACA TCT GGG GAG AGA GAT ACT AAG AGC ATA CAA 1958
Ile-Gln-Thr-Glu-Pro-Thr-Ala-Ser-Val-Ser-Ser-Gly-Asn-Glu- ATT CAG ACA GAA CCA ACC GCT TCA GTT AGC TCT GGA AAC GAG 2000Ile-Gln-Thr-Glu-Pro-Thr-Ala-Ser-Val-Ser-Ser-Gly-Asn-Glu- ATT CAG ACA GAA CCA ACC GCT TCA GTT AGC TCT GGA AAC GAG 2000
Ser-Arg-His-Asp-Ser-Glu-Ser-Met-His-Asp-Pro-Asn-Asp-Lys-Ser-Arg-His-Asp-Ser-Glu-Ser-Met-His-Asp-Pro-Asn-Asp-Lys-
AGT CGG CAT GAT TCT GAG AGC ATG CAT GAT CCA AAT GAC AAG 2042AGT CGG CAT GAT TCT GAG AGC ATG CAT GAT CCA AAT GAC AAG 2042
Lys-Asp-His-Thr-Pro-Asp-His-Asp-Val-Val-Pro-Asp-Ile-Glu-Lys-Asp-His-Thr-Pro-Asp-His-Asp-Val-Val-Pro-Asp-Ile-Glu-
AAA GAT CAC ACA CCC GAT CAC GAT GTG GTC CCG GAC ATT GAG 2084AAA GAT CAC ACA CCC GAT CAC GAT GTG GTC CCG GAC ATT GAG 2084
Ser-Ser-Thr-Asp-Lys-Gly-Glu-Ile-Arg-Asp-Ile-Glu-Gly-Glu-Ser-Ser-Thr-Asp-Lys-Gly-Glu-Ile-Arg-Asp-Ile-Glu-Gly-Glu-
TCT TCT ACT GAC AAA GGA GAG ATT CGA GAT ATA GAA GGA GAA 2126TCT TCT ACT GAC AAA GGA GAG ATT CGA GAT ATA GAA GGA GAA 2126
Val-Ala-His-Gln-Val-Ala-Glu-Ser-Phe-Ser-Lys-Lys-Tyr-Lys-Val-Ala-His-Gln-Val-Ala-Glu-Ser-Phe-Ser-Lys-Lys-Tyr-Lys-
GTT GCC CAT CAG GTA GCA GAA AGC TTT TCA AAG AAA TAC AAG 2168GTT GCC CAT CAG GTA GCA GAA AGC TTT TCA AAG AAA TAC AAG 2168
Phe-Pro-Ser-Arg-Ser-Ser-Gly-Ile-Phe-Leu-Trp-Asn-Phe-Glu- TTC CCT TCT AGA TCC TCG GGA ATA TTC TTG TGG AAC TTT GAG 2210
Gln-Leu-Lys-Met-Asn-Leu-Asp-Asp-Ile-Val-Lys-Ala-Ala-Met-Phe-Pro-Ser-Arg-Ser-Ser-Gly-Ile-Phe-Leu-Trp-Asn-Phe-Glu- TTC CCT TCT AGA TCC TCG GGA ATA TTC TTG TGG AAC TTT GAG 2210 Gln-Leu-Lys-Met-Asn-Leu-Asp-Asp-Ile-Val-Lys-Ala-Ala-Met-
CAG CTT AAA ATG AAT CTA GAT GAT ATT GTG AAA GCA GCC ATG 2252CAG CTT AAA ATG AAT CTA GAT GAT ATT GTG AAA GCA GCC ATG 2252
Asn-Val-Pro-Gly-Val-Glu-Arg-Ile-Ala-Glu-Lys-Gly-Gly-Lys-Asn-Val-Pro-Gly-Val-Glu-Arg-Ile-Ala-Glu-Lys-Gly-Gly-Lys-
AAT GTA CCA GGG GTT GAA AGG ATC GCC GAA AAG GGA GGG AAG 2294AAT GTA CCA GGG GTT GAA AGG ATC GCC GAA AAG GGA GGG AAG 2294
Leu-Pro-Leu-Arg-Cys-Ile-Leu-Gly-Phe-Val-Ala-Leu-Asp-Ser-Leu-Pro-Leu-Arg-Cys-Ile-Leu-Gly-Phe-Val-Ala-Leu-Asp-Ser-
CTT CCC CTG AGA TGT ATT TTG GGG TTT GTG GCA TTG GAC TCT 2336CTT CCC CTG AGA TGT ATT TTG GGG TTT GTG GCA TTG GAC TCT 2336
Ser-Lys-Arg-Phe-Arg-Leu-Leu-Ala-Asp-Asn-Asp-Lys-Val-Ala-Ser-Lys-Arg-Phe-Arg-Leu-Leu-Ala-Asp-Asn-Asp-Lys-Val-Ala-
TCA AAG AGA TTT AGA CTT CTT GCA GAC AAT GAC AAG GTG GCA 2378TCA AAG AGA TTT AGA CTT CTT GCA GAC AAT GAC AAG GTG GCA 2378
Arg-Leu-Ile-Gln-Glu-Asp-Ile-Asn-Ser-Tyr-Met-Ala-Arg-Leu-Arg-Leu-Ile-Gln-Glu-Asp-Ile-Asn-Ser-Tyr-Met-Ala-Arg-Leu-
AGA CTC ATC CAA GAA GAT ATC AAC AGT TAC ATG GCC CGG CTC 2420AGA CTC ATC CAA GAA GAT ATC AAC AGT TAC ATG GCC CGG CTC 2420
Glu-Glu-Ala-Glu Glu-Glu-Ala-Glu
GAG GAG GCA GAG TAAAGGCTGA GAGGACCCAT AAAAGAACTC 2462 GAG GAG GCA GAG TAAAGGCTGA GAGGACCCAT AAAAGAACTC 2462
GAATTTGGCA ATCTGGTCTT 2512
GAATTTGGCA ATCTGGTCTT 2512
^ Met-Asn-Phe-Leu-Lys-Lys-Mët-Ile-Lys-Ser-Cys-Lys-Asp- AAG ATG AAT TTC CTC AAG AAA ATG ATC AAG AGC TGT AAG GAT 2554 ^ Met-Asn-Phe-Leu-Lys-Lys-Mët-Ile-Lys-Ser-Cys-Lys-Asp- AAG ATG AAT TTC CTC AAG AAA ATG ATC AAG AGC TGT AAG GAT 2554
Glu-Glu-Thr-Gln-Lys-Tyr-Pro-Ser-Ala-Ser-Ala-Pro-Pro-Asp-Glu-Glu-Thr-Gln-Lys-Tyr-Pro-Ser-Ala-Ser-Ala-Pro-Pro-Asp-
GAA GAG ACT CAG AAG TAT CCA TCA GCA TCT GCG CCT CCA GAC 2596GAA GAG ACT CAG AAG TAT CCA TCA GCA TCT GCG CCT CCA GAC 2596
Asp-Asp-Asp-Ile-Trp-Met-Pro-Pro-Pro-Glu-Tyr-Val-Pro-Leu-Asp-Asp-Asp-Ile-Trp-Met-Pro-Pro-Pro-Glu-Tyr-Val-Pro-Leu-
GAT GAT GAC ATT TGG ATG CCC CCG CCT GAG TAT GTC CCC TTA 2638GAT GAT GAC ATT TGG ATG CCC CCG CCT GAG TAT GTC CCC TTA 2638
Thr-Gln-Val-Lys-Gly-Lys-Ala-Ser-Val-Arg-Asn-Phe-Cys-Ile-Thr-Gln-Val-Lys-Gly-Lys-Ala-Ser-Val-Arg-Asn-Phe-Cys-Ile-
ACC CAG GTC AAG GGC AAG GCC AGT GTG AGA AAC TTT TGC ATT 2680ACC CAG GTC AAG GGC AAG GCC AGT GTG AGA AAC TTT TGC ATT 2680
Ser-Gly-Glu-Val-Lys-Ile-Cys-Ser-Pro-Asn-Gly-Tyr-Ser-Phe-Ser-Gly-Glu-Val-Lys-Ile-Cys-Ser-Pro-Asn-Gly-Tyr-Ser-Phe-
AGT GGA GAG GTC AAG ATA TGT AGT CCA AAC GGG TAC TCC TTC 2722AGT GGA GAG GTC AAG ATA TGT AGT CCA AAC GGG TAC TCC TTC 2722
Lys-Ile-Leu-Arg-His-Ile-Leu-Lys-Ser-Phe-Asp-Asn-Val-Tyr-Lys-Ile-Leu-Arg-His-Ile-Leu-Lys-Ser-Phe-Asp-Asn-Val-Tyr-
AAG ATA CTC AGG CAT ATT TTG AAG TCG TTT GAT AAT GTT TAC 2764AAG ATA CTC AGG CAT ATT TTG AAG TCG TTT GAT AAT GTT TAC 2764
Ser-Gly-Asn-Arg-Arg-Met-Ile-Gly-Leu-Val-Lys-Val-Val-Ile-Ser-Gly-Asn-Arg-Arg-Met-Ile-Gly-Leu-Val-Lys-Val-Val-Ile-
TCT GGG AAC AGG AGG ATG ATC GGG TTA GTC AAA GTG GTT ATC 2806TCT GGG AAC AGG AGG ATG ATC GGG TTA GTC AAA GTG GTT ATC 2806
Gly-Leu-Val-Leu-Ser-Gly-Ser-Pro-Val-Pro-Glu-Gly-Met-Asn-Gly-Leu-Val-Leu-Ser-Gly-Ser-Pro-Val-Pro-Glu-Gly-Met-Asn-
GGG CTT GTA CTT TCA GGA TCT CCA GTC CCG GAG GGC ATG AAC 2848GGG CTT GTA CTT TCA GGA TCT CCA GTC CCG GAG GGC ATG AAC 2848
Trp-Val-Tyr-Lys-Leu-Arg-Arg-Thr-Leu-Ile-Phe-Gln-Trp-Ala-Trp-Val-Tyr-Lys-Leu-Arg-Arg-Thr-Leu-Ile-Phe-Gln-Trp-Ala-
TGG GTT TAT AAA CTT CGT AGG ACC TTA ATA TTT CAG TGG GCA 2890TGG GTT TAT AAA CTT CGT AGG ACC TTA ATA TTT CAG TGG GCA 2890
Glu-Ser-His-Gly-Pro-Leu-Glu-Gly-Glu-Glu-Leu-Glu-Tyr-Ser-Glu-Ser-His-Gly-Pro-Leu-Glu-Gly-Glu-Glu-Leu-Glu-Tyr-Ser-
GAG TCT CAT GGA CCG TTG GAA GGA GAA GAG CTT GAG TAC TCA 2932GAG TCT CAT GGA CCG TTG GAA GGA GAA GAG CTT GAG TAC TCA 2932
Gln-Glu-Ile-Thr-Trp-Asp-Asp-Glu-Ala-Glu-Phe-Val-Gly-Leu-Gln-Glu-Ile-Thr-Trp-Asp-Asp-Glu-Ala-Glu-Phe-Val-Gly-Leu-
CAA GAA ATT ACA TGG GAT GAT GAG GCA GAG TTT GTA GGC CTC 2974
Gln-Ile-Arg-Val-Ser-Ala-Arg-Gln-Cys-His-Ile-Gln-Gly-Arg- CAA ATC AGA GTG AGC GCC AGA CAA TGT CAC ATC CAG GGT CGT 3016CAA GAA ATT ACA TGG GAT GAT GAG GCA GAG TTT GTA GGC CTC 2974 Gln-Ile-Arg-Val-Ser-Ala-Arg-Gln-Cys-His-Ile-Gln-Gly-Arg- CAA ATC AGA GTG AGC GCC AGA CAA TGT CAC ATC CAG GGT CGT 3016
Leu-Trp-Cys-Ile-Asn-Met-Asn-Ser-Arg-Ala-Cys-Gln-Leu-Trp-Leu-Trp-Cys-Ile-Asn-Met-Asn-Ser-Arg-Ala-Cys-Gln-Leu-Trp-
CTC TGG TGC ATT AAC ATG AAC TCA AGA GCA TGT CAA TTA TGG 3058CTC TGG TGC ATT AAC ATG AAC TCA AGA GCA TGT CAA TTA TGG 3058
Ala-Asp-Met-Ile-Leu-Gln-Thr-Gln-Gln-Ser-Pro-Asp-Asp-Glu-Ala-Asp-Met-Ile-Leu-Gln-Thr-Gln-Gln-Ser-Pro-Asp-Asp-Glu-
GCC GAT ATG ATC TTG CAG ACC CAA CAG TCC CCG GAT GAT GAA 3100GCC GAT ATG ATC TTG CAG ACC CAA CAG TCC CCG GAT GAT GAA 3100
Asn-Thr-Ser-Leu-Leu-Leu-Glu Asn-Thr-Ser-Leu-Leu-Leu-Glu
AAC ACC TCA CTT TTA TTA GAG TAGACTCTAG CCTGTAGCTT 3141 AAC ACC TCA CTT TTA TTA GAG TAGACTCTAG CCTGTAGCTT 3141
TGCCTCTTAA TTGTTACCTC TGTTTGGAGT AGAGAAAAAC CGCGAGCAAT 3191TGCCTCTTAA TTGTTACCTC TGTTTGGAGT AGAGAAAAAC CGCGAGCAAT 3191
AGAACAATTA CCGCAACGGT GCCCGCTTTC AGCACAATAC ATATAAGCTA 3241 ACCACTGGTT TGTCTTCCTA TTCAGGGTCG AGCGAAAACG
3291
Met-Asn-Ile-Pro-AGAACAATTA CCGCAACGGT GCCCGCTTTC AGCACAATAC ATATAAGCTA 3241 ACCACTGGTT TGTCTTCCTA TTCAGGGTCG AGCGAAAACG 3291 Met-Asn-Ile-Pro-
TACATAAAAA GGCA TCTCCCT GCCATC ATG AAT ATA CCT 3339
TACATAAAAA GGCA TCTCCCT GCCATC ATG AAT ATA CCT 3339
Cys-Phe-Val-Val-Ile-Leu-Ser-Leu-Ala-Thr-Thr-His-Ser-Leu-Cys-Phe-Val-Val-Ile-Leu-Ser-Leu-Ala-Thr-Thr-His-Ser-Leu-
TGC TTT GTT GTG ATT CTC AGC TTA GCC ACT ACA CAT TCT CTG 3381TGC TTT GTT GTG ATT CTC AGC TTA GCC ACT ACA CAT TCT CTG 3381
Gly-Glu-Phe-Pro-Leu-Tyr-Thr-Ile-Pro-Glu-Lys-Ile-Glu-Lys-Gly-Glu-Phe-Pro-Leu-Tyr-Thr-Ile-Pro-Glu-Lys-Ile-Glu-Lys-
GGA GAA TTC CCC TTG TAC ACA ATT CCT GAG AAG ATA GAG AAA 3423GGA GAA TTC CCC TTG TAC ACA ATT CCT GAG AAG ATA GAG AAA 3423
Trp-Thr-Pro-Ile-Asp-Met-Ile-His-Leu-Ser-Cys-Pro-Asn-Asn-Trp-Thr-Pro-Ile-Asp-Met-Ile-His-Leu-Ser-Cys-Pro-Asn-Asn-
TGG ACT CCC ATA GAC ATG ATC CAT CTG AGT TGC CCC AAC AAC 3465TGG ACT CCC ATA GAC ATG ATC CAT CTG AGT TGC CCC AAC AAC 3465
Leu-Leu-Ser-Glu-Glu-Glu-Gly-Cys-Asn-Ala-Glu-Ser-Ser-Phe-Leu-Leu-Ser-Glu-Glu-Glu-Gly-Cys-Asn-Ala-Glu-Ser-Ser-Phe-
CTA TTA TCT GAG GAA GAA GGT TGC AAT GCA GAG TCA TCC TTT 3507CTA TTA TCT GAG GAA GAA GGT TGC AAT GCA GAG TCA TCC TTT 3507
Thr-Tyr-Phe-Glu-Leu-Lys-Ser-Gly-Tyr-Leu-Ala-His-Gln-Lys-Thr-Tyr-Phe-Glu-Leu-Lys-Ser-Gly-Tyr-Leu-Ala-His-Gln-Lys-
ACT TAC TTT GAG CTC AAG AGT GGT TAC CTA GCT CAT CAG AAG 3549 Val-Pro-Gly-Phe-Thr-Cys-Thr-Gly-Val-Val-Asn-Glu-Ala-Glu-ACT TAC TTT GAG CTC AAG AGT GGT TAC CTA GCT CAT CAG AAG 3549 Val-Pro-Gly-Phe-Thr-Cys-Thr-Gly-Val-Val-Asn-Glu-Ala-Glu-
GTT CCA GGG TTT ACC TGT ACC GGG GTC GTG AAC GAG GCA GAG 3591GTT CCA GGG TTT ACC TGT ACC GGG GTC GTG AAC GAG GCA GAG 3591
Thr-Tyr-Thr-Asn-Phe-Val-Gly-Tyr-Val-Thr-Thr-Thr-Phe-Lys-Thr-Tyr-Thr-Asn-Phe-Val-Gly-Tyr-Val-Thr-Thr-Thr-Phe-Lys-
ACA TAT ACA AAC TTC GTC GGG TAC GTC ACC ACA ACC TTC AAA 3633ACA TAT ACA AAC TTC GTC GGG TAC GTC ACC ACA ACC TTC AAA 3633
Arg-Lys-His-Phe-Arg-Pro-Thr-Val-Ala-Ala-Cys-Arg-Asp-Ala-Arg-Lys-His-Phe-Arg-Pro-Thr-Val-Ala-Ala-Cys-Arg-Asp-Ala-
AGG AAG CAC TTT AGG CCT ACA GTA GCC GCC TGT CGT GAT GCC 3675AGG AAG CAC TTT AGG CCT ACA GTA GCC GCC TGT CGT GAT GCC 3675
Tyr-Asn-Trp-Lys-Val-Ser-Gly-Asp-Pro-Arg-Tyr-Glu-Glu-Ser-Tyr-Asn-Trp-Lys-Val-Ser-Gly-Asp-Pro-Arg-Tyr-Glu-Glu-Ser-
TAC AAC TGG AAA GTG TCA GGA GAC CCC AGG TAC GAA GAG TCA 3717TAC AAC TGG AAA GTG TCA GGA GAC CCC AGG TAC GAA GAG TCA 3717
Leu-His-Thr-Pro-Tyr-Pro-Asp-Ser-Ser-Trp-Leu-Arg-Thr-Val-Leu-His-Thr-Pro-Tyr-Pro-Asp-Ser-Ser-Trp-Leu-Arg-Thr-Val-
CTC CAC ACT CCT TAT CCT GAC AGC AGT TGG TTG AGG ACT GTG 3759 Thr-Thr-Thr-Lys-Glu-Ser-Leu-Leu-Ile-Ile-Ser-Pro-Ser-Ile-CTC CAC ACT CCT TAT CCT GAC AGC AGT TGG TTG AGG ACT GTG 3759 Thr-Thr-Thr-Lys-Glu-Ser-Leu-Leu-Ile-Ile-Ser-Pro-Ser-Ile-
ACT ACA ACC AAA GAA TCA CTT CTC ATA ATA TCG CCC AGC ATC 3801
Val-Glu-Met-Asp-Ile-Tyr-Gly-Arg-Thr-Leu-His-Ser-Pro-Met-ACT ACA ACC AAA GAA TCA CTT CTC ATA ATA TCG CCC AGC ATC 3801 Val-Glu-Met-Asp-Ile-Tyr-Gly-Arg-Thr-Leu-His-Ser-Pro-Met-
GTG GAA ATG GAT ATT TAC GGC AGG ACT CTC CAT TCC CCC ATG 3843GTG GAA ATG GAT ATT TAC GGC AGG ACT CTC CAT TCC CCC ATG 3843
Phe-Pro-Ser-Gly-Val-Cys-Ser-Asn-Val-Tyr-Pro-Ser-Val-Pro-Phe-Pro-Ser-Gly-Val-Cys-Ser-Asn-Val-Tyr-Pro-Ser-Val-Pro-
TTT CCT TCA GGA GTA TGT TCC AAC GTA TAT CCC TCT GTC CCA 3885TTT CCT TCA GGA GTA TGT TCC AAC GTA TAT CCC TCT GTC CCA 3885
Ser-Cys-Glu-Thr-Asn-His-Asp-Tyr-Thr-Leu-Trp-Leu-Pro-Glu-Ser-Cys-Glu-Thr-Asn-His-Asp-Tyr-Thr-Leu-Trp-Leu-Pro-Glu-
TCC TGT GAG ACT AAT CAT GAT TAC ACA TTA TGG CTG CCT GAA 3927TCC TGT GAG ACT AAT CAT GAT TAC ACA TTA TGG CTG CCT GAA 3927
Asp-Pro-Ser-Leu-Ser-Leu-Val-Cys-Asp-Ile-Phe-Thr-Ser-Ser-Asp-Pro-Ser-Leu-Ser-Leu-Val-Cys-Asp-Ile-Phe-Thr-Ser-Ser-
GAT CCT AGT TTG AGT TTG GTC TGT GAT ATC TTT ACT TCC AGC 3969GAT CCT AGT TTG AGT TTG GTC TGT GAT ATC TTT ACT TCC AGC 3969
Asn-Gly-Lys-Lys-Ala-Met-Asn-Gly-Ser-Arg-Ile-Cys-Gly-Phe- AAC GGA AAG AAG GCC ATG AAC GGG TCA CGC ATC TGC GGA TTC 4011Asn-Gly-Lys-Lys-Ala-Met-Asn-Gly-Ser-Arg-Ile-Cys-Gly-Phe- AAC GGA AAG AAG GCC ATG AAC GGG TCA CGC ATC TGC GGA TTC 4011
Lys-Asp-Glu-Arg-Gly-Phe-Tyr-Arg-Ser-Leu-Lys-Gly-Ala-Cys-Lys-Asp-Glu-Arg-Gly-Phe-Tyr-Arg-Ser-Leu-Lys-Gly-Ala-Cys-
AAG GAT GAA AGG GGA TTC TAC AGA TCT TTA AAG GGC GCT TGC 4053AAG GAT GAA AGG GGA TTC TAC AGA TCT TTA AAG GGC GCT TGC 4053
Lys-Leu-Thr-Leu-Cys-Gly-Arg-Pro-Gly-Ile-Arg-Leu-Phe-Asp-Lys-Leu-Thr-Leu-Cys-Gly-Arg-Pro-Gly-Ile-Arg-Leu-Phe-Asp-
AAG CTG ACA TTG TGT GGA AGA CCT GGA ATT AGG TTA TTC GAC 4095 Gly-Thr-Trp-Val-Ser-Phe-Thr-Lys-Pro-Asp-Val-His-Val-Trp-AAG CTG ACA TTG TGT GGA AGA CCT GGA ATT AGG TTA TTC GAC 4095 Gly-Thr-Trp-Val-Ser-Phe-Thr-Lys-Pro-Asp-Val-His-Val-Trp-
GGA ACT TGG GTC TCT TTT ACA AAG CCG GAC GTG CAC GTA TGG 4137GGA ACT TGG GTC TCT TTT ACA AAG CCG GAC GTG CAC GTA TGG 4137
Cys-Thr-Pro-Asn-Gln-Leu-Ile-Asn-Ile-His-Asn-Asp-Arg-Leu-Cys-Thr-Pro-Asn-Gln-Leu-Ile-Asn-Ile-His-Asn-Asp-Arg-Leu-
TGC ACT CCC AAC CAA TTG ATC AAT ATA CAC AAT GAC AGA CTA 4179TGC ACT CCC AAC CAA TTG ATC AAT ATA CAC AAT GAC AGA CTA 4179
Asp-Glu-Ile-Glu-His-Leu-Ile-Val-Glu-Asp-Ile-Ile-Lys-Lys-Asp-Glu-Ile-Glu-His-Leu-Ile-Val-Glu-Asp-Ile-Ile-Lys-Lys-
GAT GAG ATA GAA CAC CTG ATC GTG GAA GAC ATC ATA AAG AAA 4221GAT GAG ATA GAA CAC CTG ATC GTG GAA GAC ATC ATA AAG AAA 4221
Arg-Glu-Glu-Cys-Leu-Asp-Thr-Leu-Glu-Thr-Ile-Leu-Met-Ser-Arg-Glu-Glu-Cys-Leu-Asp-Thr-Leu-Glu-Thr-Ile-Leu-Met-Ser-
AGA GAA GAG TGC TTA GAC ACC CTG GAA ACA ATA CTT ATG TCT 4263AGA GAA GAG TGC TTA GAC ACC CTG GAA ACA ATA CTT ATG TCT 4263
Gln-Ser-Val-Ser-Phe-Arg-Arg-Leu-Ser-His-Phe-Arg-Lys-Leu-Gln-Ser-Val-Ser-Phe-Arg-Arg-Leu-Ser-His-Phe-Arg-Lys-Leu-
CAA TCT GTT AGC TTT AGA AGG TTG AGC CAT TTC CGA AAG TTA 4305 Val-Pro-Gly-Tyr-Gly-Lys-Ala-Tyr-Thr-Ile-Leu-Asn-Gly-Ser-CAA TCT GTT AGC TTT AGA AGG TTG AGC CAT TTC CGA AAG TTA 4305 Val-Pro-Gly-Tyr-Gly-Lys-Ala-Tyr-Thr-Ile-Leu-Asn-Gly-Ser-
GTT CCA GGA TAT GGG AAG GCC TAC ACT ATT TTA AAC GGC AGC 4347GTT CCA GGA TAT GGG AAG GCC TAC ACT ATT TTA AAC GGC AGC 4347
Leu-Met-Glu-Thr-Asn-Val-Tyr-Tyr-Lys-Arg-Val-Asp-Lys-Trp-Leu-Met-Glu-Thr-Asn-Val-Tyr-Tyr-Lys-Arg-Val-Asp-Lys-Trp-
CTG ATG GAA ACA AAT GTC TAC TAC AAA AGG GTC GAC AAG TGG 4389CTG ATG GAA ACA AAT GTC TAC TAC AAA AGG GTC GAC AAG TGG 4389
Ala-Asp-Ile-Leu-Pro-Ser-Lys-Gly-Cys-Leu-Lys-Val-Gly-Gln-Ala-Asp-Ile-Leu-Pro-Ser-Lys-Gly-Cys-Leu-Lys-Val-Gly-Gln-
GCT GAC ATC TTA CCC TCT AAG GGA TGT CTG AAA GTC GGG CAA 4431GCT GAC ATC TTA CCC TCT AAG GGA TGT CTG AAA GTC GGG CAA 4431
Gln-Cys-Met-Glu-Pro-Val-Lys-Gly-Val-Leu-Phe-Asn-Gly-Ile-Gln-Cys-Met-Glu-Pro-Val-Lys-Gly-Val-Leu-Phe-Asn-Gly-Ile-
CAA TGC ATG GAA CCT GTC AAA GGA GTC CTC TTC AAT GGG ATT 4473CAA TGC ATG GAA CCT GTC AAA GGA GTC CTC TTC AAT GGG ATT 4473
Ile-Lys-Gly-Pro-Asp-Gly-Gln-Ile-Leu-Ile-Pro-Glu-Met-Gln-Ile-Lys-Gly-Pro-Asp-Gly-Gln-Ile-Leu-Ile-Pro-Glu-Met-Gln-
ATC AAG GGC CCG GAT GGC CAA ATT TTG ATC CCC GAG ATG CAG 4515 Ser-Glu-Gln-Leu-Lys-Gln-His-Met-Asp-Leu-Leu-Lys-Ala-Ala-ATC AAG GGC CCG GAT GGC CAA ATT TTG ATC CCC GAG ATG CAG 4515 Ser-Glu-Gln-Leu-Lys-Gln-His-Met-Asp-Leu-Leu-Lys-Ala-Ala-
TCA GAG CAG CTA AAG CAG CAT ATG GAC CTG TTG AAG GCG GCT 4557
Val-Phe-Pro-Leu-Arg-His-Pro-Leu-Ile-Ser-Arg-Glu-Ala-Val- GTG TTT CCT CTC CGA CAC CCT TTA ATC AGC CGG GAG GCA GTC 4599TCA GAG CAG CTA AAG CAG CAT ATG GAC CTG TTG AAG GCG GCT 4557 Val-Phe-Pro-Leu-Arg-His-Pro-Leu-Ile-Ser-Arg-Glu-Ala-Val- GTG TTT CCT CTC CGA CAC CCT TTA ATC AGC CGG GAG GCA GTC 4599
Phe-Lys-Lys-Asp-Gly-Asp-Ala-Asp-Asp-Phe-Val-Asp-Leu-His-Phe-Lys-Lys-Asp-Gly-Asp-Ala-Asp-Asp-Phe-Val-Asp-Leu-His-
TTT AAG AAA GAC GGG GAT GCC GAT GAT TTT GTG GAT CTC CAT 4641TTT AAG AAA GAC GGG GAT GCC GAT GAT TTT GTG GAT CTC CAT 4641
Met-Pro-Asp-Val-His-Lys-Ser-Val-Ser-Asp-Val-Asp-Leu-Gly-Met-Pro-Asp-Val-His-Lys-Ser-Val-Ser-Asp-Val-Asp-Leu-Gly-
ATG CCT GAT GTC CAC AAG TCT GTG TCA GAT GTC GAC CTG GGT 4683ATG CCT GAT GTC CAC AAG TCT GTG TCA GAT GTC GAC CTG GGT 4683
Leu-Pro-His-Trp-Gly-Phe-Trp-Met-Leu-Ile-Gly-Ala-Thr-Ile-Leu-Pro-His-Trp-Gly-Phe-Trp-Met-Leu-Ile-Gly-Ala-Thr-Ile-
CTG CCT CAT TGG GGT TTC TGG ATG TTG ATC GGG GCA ACA ATA 4725CTG CCT CAT TGG GGT TTC TGG ATG TTG ATC GGG GCA ACA ATA 4725
Val-Ala-Phe-Val-Val-Leu-Val-Cys-Leu-Leu-Arg-Val-Cys-Cys-Val-Ala-Phe-Val-Val-Leu-Val-Cys-Leu-Leu-Arg-Val-Cys-Cys-
GTA GCA TTT GTG GTC TTG GTA TGT TTA CTC CGT GTA TGT TGT 4767GTA GCA TTT GTG GTC TTG GTA TGT TTA CTC CGT GTA TGT TGT 4767
Lys-Arg-Val-Arg-Arg-Arg-Arg-Ser-Gly-Arg-Ala-Thr-Gln-Glu-Lys-Arg-Val-Arg-Arg-Arg-Arg-Ser-Gly-Arg-Ala-Thr-Gln-Glu-
AAG AGA GTG AGG AGG AGA AGA TCA GGA CGT GCA ACT CAG GAG 4809AAG AGA GTG AGG AGG AGA AGA TCA GGA CGT GCA ACT CAG GAG 4809
Ile-Pro-Leu-Ser-Phe-Pro-Ser-Ala-Pro-Val-Pro-Arg-Ala-Lys-Ile-Pro-Leu-Ser-Phe-Pro-Ser-Ala-Pro-Val-Pro-Arg-Ala-Lys-
ATC CCC CTG AGC TTT CCC TCT GGC CCT GTT CCT CGA GCC AAA 4851 Val-Val-Ser-Ser-Trp-Glu-Ser-Tyr-Lys-Gly-Leu-Pro-Gly-Thr ATC CCC CTG AGC TTT CCC TCT GGC CCT GTT CCT CGA GCC AAA 4851 Val-Val-Ser-Ser-Trp-Glu-Ser-Tyr-Lys-Gly-Leu-Pro-Gly-Thr
GTG GTG TCA TCT TGG GAG TCC TAT AAA GGG CTT CCA GGT ACA 4893 GTG GTG TCA TCT TGG GAG TCC TAT AAA GGG CTT CCA GGT ACA 4893
TGAAACCTTC ATCAGATTGC CTAACATATC CCCCACAACC GGATTACCTG 4943TGAAACCTTC ATCAGATTGC CTAACATATC CCCCACAACC GGATTACCTG 4943
CCTCGGCAAG ACACAACTTG ATCACATGGT GTCAAATCTC CTTTCAAACC 4993CCTCGGCAAG ACACAACTTG ATCACATGGT GTCAAATCTC CTTTCAAACC 4993
CTCCAGTGTA TAATGATTAG AGGAGGGTTG CTTGTCAATC AGGGGGTGGT 5043CTCCAGTGTA TAATGATTAG AGGAGGGTTG CTTGTCAATC AGGGGGTGGT 5043
GTTGTCTCAT ACATTCCGTT ACTCGTAAGT TGAAATCTCT CCTTTCTCAT 5093GTTGTCTCAT ACATTCCGTT ACTCGTAAGT TGAAATCTCT CCTTTCTCAT 5093
TGTCTAAATA CTTCTGAACA CAATCTCTCA ACGATTAGGT CTTCTGGTTT 5143TGTCTAAATA CTTCTGAACA CAATCTCTCA ACGATTAGGT CTTCTGGTTT 5143
TTATAAAGAG TTGCCTTCTA AAATGGGCAC TCTATAGAGC CTTCAATCTT 5193 TTTGAGGTGC GGCAATATTA GCTTGAAATA ACCTTAAGGT CTAATTTCTC 5243TTATAAAGAG TTGCCTTCTA AAATGGGCAC TCTATAGAGC CTTCAATCTT 5193 TTTGAGGTGC GGCAATATTA GCTTGAAATA ACCTTAAGGT CTAATTTCTC 5243
CTGTTTCCCA ATAATATCAC AGGAGTATCT AATTGTTCTG TGTGATGACA 5293CTGTTTCCCA ATAATATCAC AGGAGTATCT AATTGTTCTG TGTGATGACA 5293
GGACGCAATA TGATGTCTCT TCTTCTTGTA GAGTGTTGAT TCGTCAGATT 5343 GTCACCCTAG ACTGTCACAT ATGAGATTAT TGATG CATGCC 5393 iï
GGACGCAATA TGATGTCTCT TCTTCTTGTA GAGTGTTGAT TCGTCAGATT 5343 GTCACCCTAG ACTGTCACAT ATGAGATTAT TGATG CATGCC 5393 iï
* *
CCTTGGTCAA AGTCAACGCC TCCTACTTCA GTTGCAACC 5442
CCTTGGTCAA AGTCAACGCC TCCTACTTCA GTTGCAACC 5442
Met-Met-Asp-Val-Thr-Glu-Val-Tyr-Asp-Asp-Pro-Ile-Asp-Pro- Met-Met-Asp-Val-Thr-Glu-Val-Tyr-Asp-Asp-Pro-Ile-Asp-Pro-
ATG ATG GAC GTT ACG GAG GTG TAT GAC GAC CCG ATA GAC CCT 5484ATG ATG GAC GTT ACG GAG GTG TAT GAC GAC CCG ATA GAC CCT 5484
Val-Glu-Pro-Glu-Gly-Glu-Trp-Asn-Ser-Ser-Pro-Val-Val-Pro-Val-Glu-Pro-Glu-Gly-Glu-Trp-Asn-Ser-Ser-Pro-Val-Val-Pro-
GTT GAG CCA GAA GGA GAA TGG AAT AGC AGT CCC GTA GTT CCA 5526 GTT GAG CCA GAA GGA GAA TGG AAT AGC AGT CCC GTA GTT CCA 5526
AA---------------------------------------------------------------------------------------------------- 5580
- - Met-Ile-Gln-Trp-Leu-Thr-Ser-Gly-Asn-Arg-Pro- -------TA ATG ATT CAG TGG CTA ACA TCC GGG AAT AGA CCC 5622AA ------------------------------------------------- ---------------------------------------------------------- - 5580 - - Met-Ile-Gln-Trp-Leu-Thr-Ser-Gly-Asn-Arg-Pro- ------- TA ATG ATT CAG TGG CTA ACA TCC GGG AAT AGA CCC 5622
Ser-Arg-Met-...-Val-Thr-Glu-Asn-Thr-Thr-Arg-Ser-Tyr-Lys-Ser-Arg-Met -...- Val-Thr-Glu-Asn-Thr-Thr-Arg-Ser-Tyr-Lys-
TCG AGA ATG AA- GTC ACA GAG AAC ACA ACC AGG TCT TAC AAA 5664TCG AGA ATG AA- GTC ACA GAG AAC ACA ACC AGG TCT TAC AAA 5664
Val-Leu-Arg-Ala-Leu-Phe-Lys-Gly-Val-Asp-Ile-Ala-Thr-Ile-Val-Leu-Arg-Ala-Leu-Phe-Lys-Gly-Val-Asp-Ile-Ala-Thr-Ile-
GTC TTG AGA GCA CTT TTC AAG GGA GTG GAT ATA GCA ACA ATA 5706GTC TTG AGA GCA CTT TTC AAG GGA GTG GAT ATA GCA ACA ATA 5706
Lys-Ile-Gly-Gly-Val-Gly-Ala-Gln-Ala-Met-Met-Gly-Leu-Trp-Lys-Ile-Gly-Gly-Val-Gly-Ala-Gln-Ala-Met-Met-Gly-Leu-Trp-
AAA ATA GGG GGT GTG GGA GCT CAG GCA ATG ATG GGG CTG TGG 5748AAA ATA GGG GGT GTG GGA GCT CAG GCA ATG ATG GGG CTG TGG 5748
Val-Leu-Gly-Ser-His-Ser-Glu-Ser-Ser-Arg-Ser-Arg-Lys-Cys-Val-Leu-Gly-Ser-His-Ser-Glu-Ser-Ser-Arg-Ser-Arg-Lys-Cys-
GTC TTG GGG TCT CAC TCA GAA TCG TCT CGA AGC AGA AAG TGT 5790GTC TTG GGG TCT CAC TCA GAA TCG TCT CGA AGC AGA AAG TGT 5790
Leu-Ala-Asp-Leu-Ser-Ala-Phe-Tyr-Gln-Arg-Thr-Leu-Pro-Ile-Leu-Ala-Asp-Leu-Ser-Ala-Phe-Tyr-Gln-Arg-Thr-Leu-Pro-Ile-
CTA GCT GAC TTG TCT GCA TTT TAT CAG AGG ACC CTA CCT ATA 5832CTA GCT GAC TTG TCT GCA TTT TAT CAG AGG ACC CTA CCT ATA 5832
Glu-Ser-Ile-Leu-Asn-Gln-His-Leu-Asn-Glu-Gln-Arg-Thr-Thr-Glu-Ser-Ile-Leu-Asn-Gln-His-Leu-Asn-Glu-Gln-Arg-Thr-Thr-
GAG TCC ATC TTG AAC CAA CAC CTT AAT GAA CAG AGG ACT ACA 5874 Asp-Pro-Arg-Glu-Gly-Val-Leu-Ser-Gly-Leu-Asn-Arg-Val-Ser- GAC CCT AGA GAA GGA GTT TTA TCC GGA TTG AAT AGA GTT AGC 5916GAG TCC ATC TTG AAC CAA CAC CTT AAT GAA CAG AGG ACT ACA 5874 Asp-Pro-Arg-Glu-Gly-Val-Leu-Ser-Gly-Leu-Asn-Arg-Val-Ser- GAC CCT AGA GAA GGA GTT TTA TCC GGA TTG AAT AGA GTT AGC 5916
Tyr-Asp-Gln-Ser-Phe-Gly-Arg-Tyr-Leu-Gly-Asn-Leu-Tyr-Ser Tyr-Asp-Gln-Ser-Phe-Gly-Arg-Tyr-Leu-Gly-Asn-Leu-Tyr-Ser
TAT GAT CAG TCC TTT GGC CGG TAT TTA GGC AAT TTG TAC TCC 5958TAT GAT CAG TCC TTT GGC CGG TAT TTA GGC AAT TTG TAC TCC 5958
Ser-Tyr-Leu-Leu-Phe-His-Val-Ile-Ile-Leu-Tyr-Met-Asn-Ala-Ser-Tyr-Leu-Leu-Phe-His-Val-Ile-Ile-Leu-Tyr-Met-Asn-Ala-
TCT TAT CTC CTC TTT CAC GTC ATC ATA TTG TAC ATG AAT GCG 6000TCT TAT CTC CTC TTT CAC GTC ATC ATA TTG TAC ATG AAT GCG 6000
Leu-Asp-Trp-Glu-Glu- - -Thr-Ile-Leu-Ala-Leu-Trp-Arg-Leu-Asp-Trp-Glu-Glu- - -Thr-Ile-Leu-Ala-Leu-Trp-Arg-
TTG GAT TGG GAA GAG GA AG ACC ATT CTG GCC CTG TGG AGA 6042TTG GAT TGG GAA GAG GA AG ACC ATT CTG GCC CTG TGG AGA 6042
Asp-Ile-Thr-Ser-Ile-Asp-Ile-Lys-Asn-Asp-Arg-Val-Tyr-Phe-Asp-Ile-Thr-Ser-Ile-Asp-Ile-Lys-Asn-Asp-Arg-Val-Tyr-Phe-
GAC ATA ACA TCT ATA GAT ATC AAA AAT GAC CGA GTC TAC TTT 6084 Lys-Asp-Pro-Leu-Trp-Gly-Lys-Leu-Leu-Val-Thr-Lys-Asp-Phe-GAC ATA ACA TCT ATA GAT ATC AAA AAT GAC CGA GTC TAC TTT 6084 Lys-Asp-Pro-Leu-Trp-Gly-Lys-Leu-Leu-Val-Thr-Lys-Asp-Phe-
AAG GAC CCT TTG TGG GGG AAA CTC TTA GTA ACA AAA GAT TTT 6126AAG GAC CCT TTG TGG GGG AAA CTC TTA GTA ACA AAA GAT TTT 6126
Val-Tyr-Ala-His-Asn-Ser-Asn-Cys-Leu-Phe-Asp-Lys-Asn-Tyr-Val-Tyr-Ala-His-Asn-Ser-Asn-Cys-Leu-Phe-Asp-Lys-Asn-Tyr-
GTA TAT GCA CAC AAT AGC AAC TGT TTA TTT GAC AAA AAT TAC 6168GTA TAT GCA CAC AAT AGC AAC TGT TTA TTT GAC AAA AAT TAC 6168
Thr-Leu-Met-Leu-Lys-Asp-Leu-Phe-Arg-Ala-Arg-Phe-Asn-Ser-Thr-Leu-Met-Leu-Lys-Asp-Leu-Phe-Arg-Ala-Arg-Phe-Asn-Ser-
ACA CTG ATG CTA AAA GAC TTG TTC CGT GCA AGA TTC AAC TCA 6210ACA CTG ATG CTA AAA GAC TTG TTC CGT GCA AGA TTC AAC TCA 6210
Leu-Leu-Ile-Leu-Val-Ser-Pro-Pro-Asp-Ser-Arg-Tyr-Ser-Asp-Leu-Leu-Ile-Leu-Val-Ser-Pro-Pro-Asp-Ser-Arg-Tyr-Ser-Asp-
TTG CTC ATA CTT GTG TCC CCG CCG GAC TCC CGT TAC TCA GAT 6252TTG CTC ATA CTT GTG TCC CCG CCG GAC TCC CGT TAC TCA GAT 6252
Asp-Leu-Ala-Ala-Asn-Leu-Cys-Arg-Leu-Tyr-Ile-Ser-Gly-Asp-Asp-Leu-Ala-Ala-Asn-Leu-Cys-Arg-Leu-Tyr-Ile-Ser-Gly-Asp-
GAT CTG GCT GCC AAC CTG TGT CGA CTT TAC ATC TCA GGG GAT 6294 Arg-Leu-Leu-Ser-Ser-Cys-Gly-Asn-Ala-Gly-Tyr-Asp-Val-Ile-GAT CTG GCT GCC AAC CTG TGT CGA CTT TAC ATC TCA GGG GAT 6294 Arg-Leu-Leu-Ser-Ser-Cys-Gly-Asn-Ala-Gly-Tyr-Asp-Val-Ile-
AGG CTT CTC TCC AGT TGT GGG AAT GCA GGA TAT GAT GTC ATC 6336
Lys-Met-Leu-Glu-Pro-Cys-Val-Val-Asp-Leu-Leu-Val-Gln-Arg-AGG CTT CTC TCC AGT TGT GGG AAT GCA GGA TAT GAT GTC ATC 6336 Lys-Met-Leu-Glu-Pro-Cys-Val-Val-Asp-Leu-Leu-Val-Gln-Arg-
AAA ATG TTA GAG CCT TGT GTG GTG GAT CTA CTG GTT CAA AGA 6378AAA ATG TTA GAG CCT TGT GTG GTG GAT CTA CTG GTT CAA AGA 6378
Ala-Glu-Thr-Phe-Arg-Pro-Leu-Ile-His-Ser-Leu-Gly-Glu-Phe-Ala-Glu-Thr-Phe-Arg-Pro-Leu-Ile-His-Ser-Leu-Gly-Glu-Phe-
GCT GAG ACG TTC CGT CCT TTA ATT CAC TCA CTG GGG GAG TTC 6420GCT GAG ACG TTC CGT CCT TTA ATT CAC TCA CTG GGG GAG TTC 6420
Pro-Ala-Phe-Ile-Lys-Asp-Lys-Thr-Thr-Gln-Leu-Ile-Gly- CCT GCT TTC ATA AAA GAC AAA ACA ACT CAA CTG ATA GGC A-T 6462Pro-Ala-Phe-Ile-Lys-Asp-Lys-Thr-Thr-Gln-Leu-Ile-Gly- CCT GCT TTC ATA AAA GAC AAA ACA ACT CAA CTG ATA GGC A-T 6462
Phe-Gly-Pro-Cys-Asp-Tyr-Asn-Phe-Phe-Ser-Met-Leu-Gln-Asn-Phe-Gly-Pro-Cys-Asp-Tyr-Asn-Phe-Phe-Ser-Met-Leu-Gln-Asn-
TTT GGA CCA TGC GAC TAC AAT TTC TTC TCG ATG CTC CAG AAT 6504TTT GGA CCA TGC GAC TAC AAT TTC TTC TCG ATG CTC CAG AAT 6504
Phe-Asp-Asn-Ile-His-Asp-Leu-Val-Phe-Ile-Tyr-Gly-Cys-Tyr-Phe-Asp-Asn-Ile-His-Asp-Leu-Val-Phe-Ile-Tyr-Gly-Cys-Tyr-
TTC GAC AAT ATT CAT GAT TTG GTA TTT ATT TAC GGA TGT TAC 6546TTC GAC AAT ATT CAT GAT TTG GTA TTT ATT TAC GGA TGT TAC 6546
Arg-His-Trp-Gly-His-Pro-Tyr-Ile-Asp-Tyr-Arg-Lys-Gly-Leu-Arg-His-Trp-Gly-His-Pro-Tyr-Ile-Asp-Tyr-Arg-Lys-Gly-Leu-
CGG CAC TGG GGG CAT CCC TAC ATA GAC TAT AGA AAA GGG CTT 6588CGG CAC TGG GGG CAT CCC TAC ATA GAC TAT AGA AAA GGG CTT 6588
Ser-Lys-Leu-Phe-Asp-Gln-Val-His-Met-Lys-Lys-Thr-Ile-Asp-Ser-Lys-Leu-Phe-Asp-Gln-Val-His-Met-Lys-Lys-Thr-Ile-Asp-
TCC AAG CTC TTT GAT CAA GTC CAT ATG AAG AAG ACT ATA GAT 6630 Gln-Gln-Tyr-Gln-Glu-Arg-Leu-Ala-Ser-Asp-Leu-Ala-Arg-Lys-TCC AAG CTC TTT GAT CAA GTC CAT ATG AAG AAG ACT ATA GAT 6630 Gln-Gln-Tyr-Gln-Glu-Arg-Leu-Ala-Ser-Asp-Leu-Ala-Arg-Lys-
CAG CAA TAT CAA GAG CGT CTG GCT AGC GAT CTA GCC AGG AAG 6672CAG CAA TAT CAA GAG CGT CTG GCT AGC GAT CTA GCC AGG AAG 6672
Ile-Leu-Arg-Trp-Gly-Phe-Glu-Lys-Tyr-Ser-Lys-Trp-Tyr-Leu-Ile-Leu-Arg-Trp-Gly-Phe-Glu-Lys-Tyr-Ser-Lys-Trp-Tyr-Leu-
ATT CTG CGT TGG GGG TTC GAA AAG TAC TCC AAA TGG TAT CTA 6714ATT CTG CGT TGG GGG TTC GAA AAG TAC TCC AAA TGG TAT CTA 6714
Asp-Thr-Gly-Val-Ile-Pro-Lys-Asp-His-Pro-Leu-Ala-Pro-Tyr-Asp-Thr-Gly-Val-Ile-Pro-Lys-Asp-His-Pro-Leu-Ala-Pro-Tyr-
GAT ACA GGT GTC ATT CCC AAA GAC CAT CCC CTG GCT CCT TAT 6756GAT ACA GGT GTC ATT CCC AAA GAC CAT CCC CTG GCT CCT TAT 6756
Ile-Ala-Thr-Gln-Thr-Trp-Pro-Pro-Lys-His-Val-Val-Asp-Leu-Ile-Ala-Thr-Gln-Thr-Trp-Pro-Pro-Lys-His-Val-Val-Asp-Leu-
ATT GCA ACA CAG ACA TGG CCC CCG AAA CAT GTG GTG GAT CTC 6798ATT GCA ACA CAG ACA TGG CCC CCG AAA CAT GTG GTG GAT CTC 6798
Leu-Gly-Asp-Ser-Trp-His-Thr-Leu-Pro-Met-Thr-Leu-Gly-Asp-Ser-Trp-His-Thr-Leu-Pro-Met-Thr-
CTG GGA GAT TCT TGG CAC ACT CTC CCG ATG ACT----------------------6840 ----------------------------------------------------------------------------------------------- 6900 --------------------------------------------------------------------------------------------------- 6960 --------------------------------------------------------------------------------------------------- 7020 ------------------------------------------------------------------------------------------------------ 7080 ------------------------------------------------------------------------------------------------------- 7140 ------------------------------------------------------------------------------------------------------- 7200 ------------------------------------------------------------------------------------------------------- 7260 ------------------------------------------------------------------------------------------------------- 7320 ------------------------------------------------------------------------------------------------------- 7380 ------------------------------------------------------------------------------------------------------- 7440 ------------------------------------------------------------------------------------------------------- 7500 ------------------------------------------------------------------------------------------------------- 7560 ------------------------------------------------------------------------------------------------------ 7620 ------------------------------------------------------------------------------------------------------- 7680 ------------------------------------------------------------------------------------------------------- 7740 ------------------------------------------------------------------------------------------------------- 7800 ------------------------------------------------------------------------------------------------------- 7860 ------------------------------------------------------------------------------------------------------- 7920
------------------------------------------------------------------------------------------------------- 7980 ------------------------------------------------------------------------------------------------------- 8040 ------------------------------------------------------------------------------------------------------- 8100 ------------------------------------------------------------------------------------------------------- 8160 ------------------------------------------------------------------------------------------------------- 8220 ------------------------------------------------------------------------------------------------------ 8280 ------------------------------------------------------------------------------------------------------- 8340 ------------------------------------------------------------------------------------------------------- 8400 ------------------------------------------------------------------------------------------------------- 8460 ------------------------------------------------------------------------------------------------------- 8520 ------------------------------------------------------------------------------------------------------- 8580 ------------------------------------------------------------------------------------------------------- 8640 Glu-Ser-Phe-Leu-Asn-Ser-Glu-Ile-His-Gly-Ile-Asn-Arg-Val-CTG GGA GAT TCT TGG CAC ACT CTC CCG ATG ACT ---------------------- 6840 ---------------- ---------------------------------------------------------- ----------------------------- 6900 -------------------- ---------------------------------------------------------- ----------------------------- 6960 -------------------- ---------------------------------------------------------- ----------------------------- 7020 -------------------- ---------------------------------------------------------- -------------------------------- 7080 ----------------- ---------------------------------------------------------- ------------------------------------ 7140 ------------- ---------------------------------------------------------- ---------------------------------------- 7200 --------- ---------------------------------------------------------- -------------------------------------------- 7260 ----- ---------------------------------------------------------- ------------------------------------------------ 7320 - ---------------------------------------------------------- ------------------------ ---------------------------- 7380 --------------------- ---------------------------------------------------------- -------------------------------- 7440 ----------------- ---------------------------------------------------------- ------------------------------------ 7500 ------------- ---------------------------------------------------------- ---------------------------------------- 7560 --------- ---------------------------------------------------------- ------------------------------------------- 7620 ------ ---------------------------------------------------------- ----------------------------------------------- 7680 - ---------------------------------------------------------- ---------------------------------------------------------- - 7740 ------------------------------------------------ ---------------------------------------------------------- ----- 7800 -------------------------------------------- ---------------------------------------------------------- --------- 7860 ---------------------------------------- ---------------------------------------------------------- ----- -------- 7920 ---------------------------------------------------------- ---------------------------------------------------------- --- 7980 ---------------------------------------------- ---------------------------------------------------------- ------- 8040 ------------------------------------------ ---------------------------------------------------------- ----------- 8100 -------------------------------------- ---------------------------------------------------------- --------------- 8160 ---------------------------------- ---------------------------------------------------------- ------------------- 8220 ------------------------------ ---------------------------------------------------------- ---------------------- 8280 --------------------------- ---------------------------------------------------------- -------------------------- 8340 ----------------------- ---------------------------------------------------------- ------------------------------ 8400 ------------------- ---------------------------------------------------------- ---------------------------------- 8460 --------------- ----- ---------------------------------------------------------- --------------------------------- 8520 ---------------- ---------------------------------------------------------- ------------------------------------- 8580 ------------ ---------------------------------------------------------- ----------------------------------------- 8640 Glu-Ser-Phe-Leu- Asn-Ser-Glu-Ile-His-Gly-Ile-Asn-Arg-Val-
GAG TCT TTC CTT AAT TCC GAG ATC CAT GGG ATA AAC AGG GTG 8682GAG TCT TTC CTT AAT TCC GAG ATC CAT GGG ATA AAC AGG GTG 8682
Thr-Gln-Thr-Pro-Gln-Arg-Leu Thr-Gln-Thr-Pro-Gln-Arg-Leu
ACA CAA ACC CCT CAA CGA CTC -------------------------------------------8760 ACA CAA ACC CCT CAA CGA CTC ------------------------------------------- 8760
Val-Asp-Leu-Gly-Pro- ------------------------------------------------------GT- GAC CTT GGT CCC 8820 Val-Asp-Leu-Gly-Pro- ---------------------------------------- -------------- GT- GAC CTT GGT CCC 8820
Lys-Ser-Ser-Val-Ala-Cys-Gly-Cys-Tyr-Thr-Arg-Glu-Val-Gly-Lys-Ser-Ser-Val-Ala-Cys-Gly-Cys-Tyr-Thr-Arg-Glu-Val-Gly-
AAG TCC TCA GTG GCT TGT GGG TGT TAT ACC AGG GAG GTT GGA 8862AAG TCC TCA GTG GCT TGT GGG TGT TAT ACC AGG GAG GTT GGA 8862
Asn-Pro-Arg-Ile-Ser-Val-Ser-Val-Leu-Pro-Ser-Phe-Asp-Pro-Asn-Pro-Arg-Ile-Ser-Val-Ser-Val-Leu-Pro-Ser-Phe-Asp-Pro-
AAC CCC CGG ATC TCT GTC TCA GTG TTG CCT TCC TTT GAC CCT 8904AAC CCC CGG ATC TCT GTC TCA GTG TTG CCT TCC TTT GAC CCT 8904
Ser-Phe-Leu-Ser-Arg-Gly-Pro-Leu-Lys-Gly-Tyr-Leu-Gly-Ser-Ser-Phe-Leu-Ser-Arg-Gly-Pro-Leu-Lys-Gly-Tyr-Leu-Gly-Ser-
TCT TTC CTC TCA AGG GGC CCT CTT AAG GGG TAC TTA GGA TCT 8946TCT TTC CTC TCA AGG GGC CCT CTT AAG GGG TAC TTA GGA TCT 8946
Ser-Thr-Ser-Met-Ser-Thr-Gln-Leu-Phe-His-Ser-Trp-Glu-Lys-Ser-Thr-Ser-Met-Ser-Thr-Gln-Leu-Phe-His-Ser-Trp-Glu-Lys-
TCC ACA TCT ATG TCC ACT CAG TTG TTC CAC TCA TGG GAG AAA 8988TCC ACA TCT ATG TCC ACT CAG TTG TTC CAC TCA TGG GAG AAA 8988
Val-Thr-Asn-Val-His-Val-Val-Lys-Arg-Ala-Leu-Ser-Leu-Lys-Val-Thr-Asn-Val-His-Val-Val-Lys-Arg-Ala-Leu-Ser-Leu-Lys-
GTC ACA AAT GTT CAT GTG GTC AAG AGG GCT CTA TCA CTC AAA 9030GTC ACA AAT GTT CAT GTG GTC AAG AGG GCT CTA TCA CTC AAA 9030
Glu-Ser-Ile-Asn-Trp-Phe-Val-Ser-Arg-Glu-Ser-Asn-Leu-Ala-Glu-Ser-Ile-Asn-Trp-Phe-Val-Ser-Arg-Glu-Ser-Asn-Leu-Ala-
GAG TCC ATC AAC TGG TTT GTG TCT CGG GAG TCT AAC TTG GCA 9072GAG TCC ATC AAC TGG TTT GTG TCT CGG GAG TCT AAC TTG GCA 9072
Lys-Thr-Leu-Ile-Gly-Asn-Ile-Leu-Ser-Leu-Thr-Gly-Pro-Ile-Lys-Thr-Leu-Ile-Gly-Asn-Ile-Leu-Ser-Leu-Thr-Gly-Pro-Ile-
AAG ACT CTG ATA GGA AAC ATA CTG TCC CTA ACA GGA CCC ATC 9114AAG ACT CTG ATA GGA AAC ATA CTG TCC CTA ACA GGA CCC ATC 9114
Phe-Ser-Ile-Glu-Glu-Ala-Pro-Val-Phe-Lys-Arg-Thr-Gly-Ser-Phe-Ser-Ile-Glu-Glu-Ala-Pro-Val-Phe-Lys-Arg-Thr-Gly-Ser-
TTT TCC ATA GAG GAG GCT CCG GTT TTC AAG AGG ACC GGC TCA 9156TTT TCC ATA GAG GAG GCT CCG GTT TTC AAG AGG ACC GGC TCA 9156
Ala-Leu-His-Arg-Phe-Lys-Ser-Ala-Arg-Tyr-Ser-Glu-Gly-Gly-Ala-Leu-His-Arg-Phe-Lys-Ser-Ala-Arg-Tyr-Ser-Glu-Gly-Gly-
GCT TTA CAT CGA TTC AAA TCT GCT AGG TAT AGT GAG GGC GGT 9198GCT TTA CAT CGA TTC AAA TCT GCT AGG TAT AGT GAG GGC GGT 9198
Tyr-Pro-Ala-Val-Cys-Pro-Tyr-Pro-Ala-Val-Cys-Pro-
TAT CCA GCC GTG TGT CCC A----------------------------------------------------------9240 ------------------------------------------------------------------------------------------------------ 9300 ------------------------------------------------------------------------------------------------------- 9360 ------------------------------------------------------------------------------------------------------- 9420
------------------------------------------------------------------------------------------------------- 9480 ------------------------------------------------------------------------------------------------------- 9540 ------------------------------------------------------------------------------------------------------ 9600 ------------------------------------------------------------------------------------------------------- 9660 ------------------------------------------------------------------------------------------------------- 9720 ------------------------------------------------------------------------------------------------------- 9780 ------------------------------------------------------------------------------------------------------ 9840 ------------------------------------------------------------------------------------------------------ 9900 ------------------------------------------------------------------------------------------------------- 9960 ------------------------------------------------------------------------------------------------------ 10020 ------------------------------------------------------------------------------------------------------ 10080 ------------------------------------------------------------------------------------------------------ 10140 ------------------------------------------------------------------------------------------------------ 10200 ------------------------------------------------------------------------------------------------------ 10260 ------------------------------------------------------------------------------------------------------ 10320 ------------------------------------------------------------------------------------------------------ 10380 ------------------------------------------------------------------------------------------------------ 10440 ----------------------------------------------------------------------------------------------------- 10500 ---------------------------------------------------------------------------------------------------- 10560 ------------------------------------------------------------------------------------------------------ 10620 ------------------------------------------------------------------------------------------------------ 10680 ---------------------------------------------------------------------------------------------------- 10740 ------------------------------------------------------------------------------------------------------ 10800 ------------------------------------------------------------------------------------------------------ 10860 ----------------------------------------------------------------------------------------------------- 10920 ------------------------------------------------------------------------------------------------------ 10980 ------------------------------------------------------------------------------------------------------ 11040 ------------------------------------------------------------------------------------------------------ 11100 ------------------------------------------------------------------------------------------------------ 11112 ----------------------------------------------------------------------------------------------------- 11280 ------------------------------------------------------------------------------------------------------ 11340 ----------------------------------------------------------------------------------------------------- 11400 ---------------------------------------------------------------------------------------------------- 11460 ---------------------------------------------------------------------------------------------------- 11520 TAT CCA GCC GTG TGT CCC A ------------------------------------------- --------------- 9240 ---------------------------------- ---------------------------------------------------------- ------------------ 9300 ------------------------------- ---------------------------------------------------------- ---------------------- 9360 --------------------------- ---------------------------------------------------------- -------------------------- 9420 ---------------------------------------------------------- ---------------------------------------------------------- --- 9480 ---------------------------------------------- ---------------------------------------------------------- ------- 9540 ------------------------------------------ ---------------------------------------------------------- ---------- 9600 --------------------------------------- ---------------------------------------------------------- -------------- 9660 ----------------------------------- ---------------------------------------------------------- ------------------ 9720 ------------------------------- ---------------------------------------------------------- ---------------------- 9780 --------------------------- ---------------------------------------------------------- ------------------------- 9840 ------------------------ ---------------------------------------------------------- ---------------------------- 9900 --------------------- ---------------------------------------------------------- -------------------------------- 9960 ----------------- ----- ---------------------------------------------------------- ------------------------------ 10020 ------------------- ---------------------------------------------------------- --------------------------------- 10080 ---------------- ---------------------------------------------------------- ------------------------------------ 10140 ------------- ---------------------------------------------------------- --------------------------------------- 10200 ---------- ---------------------------------------------------------- ------------------------------------------ 10260 ------- ---------------------------------------------------------- --------------------------------------------- 10320 ---- ---------------------------------------------------------- ------------------------------------------------ 10380 - ---------------------------------------------------------- ---------------------------------------------------------- - 10440 ------------------------------------------------ ---------------------------------------------------------- --- 10500 ------------------------------------------ ---------------------------------------------------------- -------- 10560 ----------------------------------------- ---------------------------------------------------------- ----------- 10620 -------------------------------------- ---------------------------------------------------------- -------------- 10680 ----------------------------------- ---------------------------------------------------------- --------------- 10740 ---------------------------------- ---------------------------------------------------------- ------------------ 10800 ------------------------------- ---------------------------------------------------------- --------------------- 10860 ---------------------------- ---------------------------------------------------------- ----------------------- 10920 -------------------------- ---------------------------------------------------------- -------------------------- 10980 ----------------------- ---------------------------------------------------------- ----------------------------- 11040 -------------------- ---------------------------------------------- ------------------------------------ 11100 ------------- ---------------------------------------------------------- --------------------------------------- 11112 ---------- ---------------------------------------------------------- ----------------------------------------- 11280 -------- ---------------------------------------------------------- -------------------------------------------- 11340 ----- ---------------------------------------------------------- ---------------------------------------------- 11400 --- ---------------------------------------------------------- ----------------------------------------------- 11460 - ---------------------------------------------------------- ------------------------------------------------ 11520
Leu-Tyr-Asn-Ser-Pro-Val-Thr-Tyr-Tyr-Phe-Gly-Lys---- -AG CTC TAC AAC TCT CCT GTG ACT TAT TAC TTT GGA AAG 11562 Leu-Tyr-Asn-Ser-Pro-Val-Thr-Tyr-Tyr-Phe-Gly-Lys ---- -AG CTC TAC AAC TCT CCT GTG ACT TAT TAC TTT GGA AAG 11562
Gln-Thr-Ile-Lys-Gly-Arg-Arg-Tyr-Leu-Ser-Trp-Ser-Trp-Ala-Gln-Thr-Ile-Lys-Gly-Arg-Arg-Tyr-Leu-Ser-Trp-Ser-Trp-Ala-
CAG ACT ATC AAA GGG AGG AGG TAT CTA TCG TGG AGT TGG GCC 11604CAG ACT ATC AAA GGG AGG AGG TAT CTA TCG TGG AGT TGG GCC 11604
Asn-Ser-Ser-Pro-Ile-Phe-Lys-Lys-Val-Ala-Cys-Asn-Ser-Ser- AAC TCA AGT CCA ATC TTC AAA AAG GTG GCA TGC AAC TCC TCT 11646Asn-Ser-Ser-Pro-Ile-Phe-Lys-Lys-Val-Ala-Cys-Asn-Ser-Ser- AAC TCA AGT CCA ATC TTC AAA AAG GTG GCA TGC AAC TCC TCT 11646
Ile-Ser-Leu-Ser-Ser-His-Trp-Ile-Arg-Leu-Ile-Tyr-Lys-Ile-Ile-Ser-Leu-Ser-Ser-His-Trp-Ile-Arg-Leu-Ile-Tyr-Lys-Ile-
ATC AGT CTA .TCC TCT CAC TGG ATA AGG TTG ATA TAC AAG ATA 11688ATC AGT CTA .TCC TCT CAC TGG ATA AGG TTG ATA TAC AAG ATA 11688
Val-Lys-Thr-Thr-Arg-Leu-Asn-Cys-Ser-Pro-Arg-Asp-Met-Leu-Val-Lys-Thr-Thr-Arg-Leu-Asn-Cys-Ser-Pro-Arg-Asp-Met-Leu-
GTC AAA ACC ACT CGC CTG AAT TGC TCT CCT AGG GAC ATG TTA 11730 Arg-Glu-Thr-Glu-Ala-Cys-Leu-Arg-Thr-Tyr-Asn-Lys-Trp-Ile-GTC AAA ACC ACT CGC CTG AAT TGC TCT CCT AGG GAC ATG TTA 11730 Arg-Glu-Thr-Glu-Ala-Cys-Leu-Arg-Thr-Tyr-Asn-Lys-Trp-Ile-
AGA GAG ACA GAA GCT TGC CTT AGA ACC TAT AAC AAG TGG ATC 11772
Asn-Ile-Arg-Asp-Thr-Arg-Ser-Arg-Thr-Ser-Ile-Leu-Asp-Tyr- AAC ATA AGA GAC ACA AGA TCT AGA ACT TCG ATA TTG GAC TAC 11814AGA GAG ACA GAA GCT TGC CTT AGA ACC TAT AAC AAG TGG ATC 11772 Asn-Ile-Arg-Asp-Thr-Arg-Ser-Arg-Thr-Ser-Ile-Leu-Asp-Tyr- AAC ATA AGA GAC ACA AGA TCT AGA ACT TCG ATA TTG GAC TAC 11814
Cys-Cys-Leu
Cys-Cys-Leu
TGC. TGT CTT TAGTCTAATC AATGGTGATA GACTTGGAGA GCATCATTGA 11863
G TAGCAATTAC CTTATGCATT GTCCTGTGAT TATTTTTGAT 11913 TTTTATATGG TTTTTTTGTT AAGCGT 11939 TGC. TGT CTT TAGTCTAATC AATGGTGATA GACTTGGAGA GCATCATTGA 11863 G TAGCAATTAC CTTATGCATT GTCCTGTGAT TATTTTTGAT 11913 TTTTATATGG TTTTTTTGTT AAGCGT 11939
(I)
(I)
L'invention a également pour objet, des fragments de ladite séquence codant pour un des peptides et/ou pour un fragment peptidique du virus Mokola ainsi que des fragments de ladite séquence non codants correspondant à une zone du génome Mokola variable, c'est-à- dire non conservée d'un gène commun à tous les Lyssavirus. The subject of the invention is also, fragments of said sequence coding for one of the peptides and / or for a peptide fragment of the Mokola virus as well as fragments of said non-coding sequence corresponding to a variable region of the Mokola genome, that is to say ie not conserved of a gene common to all Lyssaviruses.
Parmi lesdits fragments codants, elle englobe entre autres : Among said coding fragments, it includes inter alia:
- un fragment qui code pour la nucléoprotéine - a fragment which codes for the nucleoprotein
N et correspond aux nucléotides 71-1420 de ladite séquence d'ADNc ; N and corresponds to nucleotides 71-1420 of said cDNA sequence;
- un fragment qui code pour la protéine M1 et correspond aux nucléotides 1524-2432 de ladite séquence d'ADNc ; a fragment which codes for the protein M1 and corresponds to nucleotides 1524-2432 of said cDNA sequence;
- un fragment qui code pour la protéine M2 et correspond aux nucléotides 2516-3121 de ladite séquence d'ADNc ; a fragment which codes for the protein M2 and corresponds to nucleotides 2516-3121 of said cDNA sequence;
- un fragment qui code pour la protéine G et correspond aux nucléotides 3328-4893 de ladite séquence d'ADNc ; - A fragment which codes for protein G and corresponds to nucleotides 3328-4893 of said cDNA sequence;
- un fragment qui code pour l'extrémité NH2 terminale de la protéine L et correspond aux nucléotides 5443-6831 de ladite séquence d'ADNc. a fragment which codes for the NH 2 terminal end of the L protein and corresponds to nucleotides 5443-6831 of said cDNA sequence.
Parmi lesdits fragments non-codants, elle englobe entre autre un fragment qui correspond aux nucléotides 4897-5442 de ladite séquence, lequel fragment correspond au pseudogène psi rabique. Among said non-coding fragments, it includes inter alia a fragment which corresponds to nucleotides 4897-5442 of said sequence, which fragment corresponds to the psi rabies pseudogen.
La présente invention a également pour objet la séquence de l'ARN genomique du virus Mokola, caractérisée en ce qu'elle comprend environ 12 000 nucléotides, en ce qu'il s'agit d'un ARN monocaténaire négatif non segmenté et non polyadénylé, en ce qu'elle présente successivement de 3' en 5' le gène codant pour l'ARN "leader" puis les gènes codant pour la nucléoprotéine N, la phosphoprotéine M1, la protéine de matrice M2, la gly
coprotéine G et la protéine polymérase L et en ce que ledit génome est toujours associé à la nucléoprotéine N. The subject of the present invention is also the sequence of the genomic RNA of the Mokola virus, characterized in that it comprises approximately 12,000 nucleotides, in that it is a non-segmented and non-polyadenylated negative single-stranded RNA, in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the nucleoprotein N, the phosphoprotein M1, the matrix protein M2, the gly co-protein G and protein polymerase L and in that said genome is always associated with nucleoprotein N.
La présente invention a également pour objet les produits de transcription du virus Mokola, caractérisés en ce qu'ils sont constitués de 5 fragments monocistroniques successifs codant à partir de l'extrémité 3' pour les protéines N, M1, M2, G et L, à savoir : The present invention also relates to the transcripts of the Mokola virus, characterized in that they consist of 5 successive monocistronic fragments coding from the 3 ′ end for the proteins N, M1, M2, G and L, to know :
- un fragment correspondant aux nucléotides 59-1484 de la séquence de formule I, associé à un poly - a fragment corresponding to nucleotides 59-1484 of the sequence of formula I, associated with a poly
A ; AT ;
- un fragment correspondant aux nucléotides 1495-2489 de la séquence de formule I, associé à un poly A ; - A fragment corresponding to nucleotides 1495-2489 of the sequence of formula I, associated with a poly A;
- un fragment correspondant aux nucléotides - a fragment corresponding to the nucleotides
2501-3283 de la séquence de formule I, associé à un poly A ; 2501-3283 of the sequence of formula I, associated with a poly A;
- un fragment correspondant aux nucléotides 3307-5380 de la séquence de formule I, associée à un poly A et codant pour la protéine G, a fragment corresponding to nucleotides 3307-5380 of the sequence of formula I, associated with a poly A and coding for the protein G,
- un fragment de grande taille codant pour la protéine L ; - a large fragment encoding the L protein;
- ainsi qu'un fragment bicistronique M1-M2. - as well as a bicistronic fragment M1-M2.
La présente invention a également pour objet des clones d'ADNc de l'ARN genomique du virus Mokola, caractérisés en ce qu'ils correspondent à l'ensemble du génome, de l'extrémité 3' à l'extrémité 5', à savoir : The present invention also relates to cDNA clones of the genomic RNA of the Mokola virus, characterized in that they correspond to the entire genome, from the 3 ′ end to the 5 ′ end, namely :
- un fragment qui mesure 4 150 nucléotides, dénommé ci-après pMD10 et correspondant à la séquence codant pour l'ARN "leader" pour la nucléoprotéine N, la protéine M1, la protéine M2 et un fragment de la séquence codant pour la protéine G ; a fragment which measures 4,150 nucleotides, hereinafter called pMD10 and corresponding to the sequence coding for RNA "leader" for nucleoprotein N, protein M1, protein M2 and a fragment of the sequence coding for protein G ;
- un fragment qui mesure 2 850 nucléotides, dénommé ci-après pMA10 et correspondant à un fragment de la séquence codant pour la protéine G et à un fragment codant pour la protéine L ;
- un fragment, réalisé par la jonction des inserts pMD10 et pMA10, au niveau d'un site BglII, qui, à partir de l'extrémité 3', comprend 6 830 nucléotides et contient la séquence codante pour l'ARN leader, les protéines N, M1, M2 et G ainsi que les 1 420 premiers nucléotides du gène L et ci-après dénommé pM7 ; a fragment which measures 2,850 nucleotides, hereinafter called pMA10 and corresponding to a fragment of the sequence coding for protein G and to a fragment coding for protein L; - a fragment, produced by the junction of the inserts pMD10 and pMA10, at a BglII site, which, from the 3 ′ end, comprises 6,830 nucleotides and contains the coding sequence for the leader RNA, the proteins N, M1, M2 and G as well as the first 1,420 nucleotides of the L gene and hereinafter called pM7;
- un fragment d'environ 3 300 nucléotides, dénommé pMB5 et correspondant à un fragment de la séquence codant pour la protéine L ; a fragment of approximately 3,300 nucleotides, called pMB5 and corresponding to a fragment of the sequence coding for the protein L;
- un fragment d'environ 2 800 nucléotides, ciaprès dénommé pM12, correspondant à un fragment de la séquence codant pour la protéine L ; - a fragment of approximately 2800 nucleotides, hereafter called pM12, corresponding to a fragment of the sequence coding for the protein L;
- un fragment d'environ 700 nucléotides, ciaprès dénommé pMR15a et correspondant à un fragment de la séquence codant pour la protéine L ainsi qu'à l'extrémité 5 ' non transcrite du génome ; - a fragment of approximately 700 nucleotides, hereinafter called pMR15a and corresponding to a fragment of the sequence coding for the protein L as well as at the 5 'non-transcribed end of the genome;
lesquels fragments, pris séparément, présentent chacun une aptitude à s'hybrider de manière spécifique à un fragment d'ARN provenant de la transcription ou de la replication du génome Mokola ou à un fragment d'ADNc. which fragments, taken separately, each have an ability to hybridize specifically to an RNA fragment originating from the transcription or replication of the Mokola genome or to a cDNA fragment.
Conformément à l'invention, le clone pM7 a été déposé sous le numéro 1-847 en date du 22 mars 1989 auprès de la Collection Nationale de cultures de microorganismes tenue par l'Institut Pasteur. In accordance with the invention, the pM7 clone was deposited under the number 1-847 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
Conformément à l'invention, le clone pMB5 a été déposé sous le numéro 1-848 en date du 22 mars 1989 auprès de la Collection Nationale de cultures de microorganismes tenue par l'Institut Pasteur. In accordance with the invention, the pMB5 clone was deposited under the number 1-848 dated March 22, 1989 with the National Collection of microorganism cultures held by the Pasteur Institute.
Conformément à l'invention, le clone pM12 a été déposé sous le numéro 1-849 en date du 22 mars 1989 auprès de la Collection Nationale de cultures de microorganismes tenue par l'Institut Pasteur. In accordance with the invention, the pM12 clone was deposited under the number 1-849 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
Conformément à l'invention, le clone pMR15a a été déposé sous le numéro 1-850 en date du 22 mars 1989 auprès de la Collection Nationale de cultures de microorganismes tenue par l'Institut Pasteur.
La présente invention a également pour objet des sondes nucléotidiques, caractérisées en ce qu'elles sont constituées par une séquence nucleotidique telle que définie ci-dessus ou un fragment de celle-ci, marquée à l'aide d'un marqueur tel qu'un isotope radioactif, une enzyme appropriée ou un fluorochrome. In accordance with the invention, the clone pMR15a was deposited under the number 1-850 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute. The present invention also relates to nucleotide probes, characterized in that they consist of a nucleotide sequence as defined above or a fragment thereof, labeled with the aid of a marker such as a radioactive isotope, a suitable enzyme or a fluorochrome.
Selon un mode de réalisation desdites sondes, on peut citer la séquence 4675-5568 ou un fragment de celle-ci et notamment le fragment 4897-5442 ou leurs brins complémentaires. According to one embodiment of said probes, mention may be made of the sequence 4675-5568 or a fragment thereof and in particular the fragment 4897-5442 or their complementary strands.
De telles sondes sont spécifiques du virus Mokola. Such probes are specific for the Mokola virus.
La présente invention a également pour objet des peptides ou fragments peptidiques, caractérisés en ce qu'ils sont codés par au moins un fragment tel que défini ci-dessus ou une portion de fragment ou une combinaison de plusieurs fragments tels que définis ci-dessus. The present invention also relates to peptides or peptide fragments, characterized in that they are encoded by at least one fragment as defined above or a fragment portion or a combination of several fragments as defined above.
Selon un mode de réalisation avantageux, ledit peptide est codé par le gène de la nucléoprotéine N et présente une séquence en acides aminés qui répond à la formule II ci-après : According to an advantageous embodiment, said peptide is encoded by the nucleoprotein N gene and has an amino acid sequence which corresponds to formula II below:
Met-Glu-Ser-Asp-Lys-Ile-Val-Phe-Lys-Val-Asn-Asn-Gln-Val- Val-Ser-Leu-Lys-Pro-Glu-Val-Ile-Ser-Asp-Gln-Tyr-Glu-Tyr- Lys-Tyr-Pro-Ala-Ile-Leu-Asp-Gly-Lys-Lys-Pro-Gly-Ile-Thr- Leu-Gly-Lys-Ala-Pro-Asp-Leu-Asn-Thr-Ala-Tyr-Lys-Ser-Ile- Leu-Ser-Gly-Met-Lys-Ala-Ala-Lys-Leu-Asp-Pro-Asp-Asp-Val- Cys-Ser-Tyr-Leu-Ala-Ala-Ala-Met-His-Leu-Phe-Glu-Gly-Val- Cys-Pro-Glu-Asp-Trp-Val-Ser-Tyr-Gly-Ile-Val-Ile-Ala-Lys- Lys-Gly-Glu-Lys-Ile-Asn-Pro-Ser-Val-Ile-Val-Asp-Ile-Val- Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp-Ala-Gln-Ala-Gly-Gly-Thr- Asp-Val-Ile-Arg-Asp-Pro-Thr-Met-Ala-Glu-His-Ala-Ser-Leu- Val-Gly-Leu-Leu-Leu-Cys-Leu-Tyr-Arg-Leu-Ser-Lys-Ile-Val- Gly-Gln-Asn-Thr-Ala-Asn-Tyr-Lys-Thr-Asn-Val-Ala-Asp-Arg- Met-Glu-Gln-Ile-Phe-Glu-Thr-Ala-Pro-Phe-Ala-Lys-Val-Val- Glu-His-His-Thr-Leu-Met-Thr-Thr-His-Lys-Met-Cys-Ala-Asn- Trp-Ser-Thr-Ile-Pro-Asn-Phe-Arg-Phe-Leu-Val-Gly-Thr-Tyr- Asp-Met-Phe-Phe-Ala-Arg-Val-Glu-His-Ile-Tyr-Ser-Ala-Leu- Arg-Val-Gly-Thr-Val-Val-Thr-Ala-Tyr-Glu-Asp-Cys-Ser-Gly- Leu-Val-Ser-Phe-Thr-Gly-Phe-Ile-Lys-Gln-Ile-Asn-Leu-Ser- Pro-Arg-Asp-Ala-Leu-Leu-Tyr-Phe-Phe-His-Lys-Asn-Phe-Glu- Gly-Glu-Ile-Lys-Arg-Met-Phe-Glu-Pro-Gly-Gln-Glu-Thr-Ala- Val-Pro-His-Ser-Tyr-Phe-Ile-His-Phe-Arg-Ala-Leu-Gly-Leu-
Ser-Gly-Lys-Ser-Pro-Tyr-Ser-Ser-Asn-Ala-Val-Gly-His-Thr- Phe-Asn-Leu-Ile-His-Phe-Val-Gly-Cys-Tyr-Met-Gly-Gln-Ile- Arg-Ser-Leu-Asn-Ala-Thr-Val-Ile-Gln-Thr-Cys-Ala-Pro-Leu- Lys-Gly-Ala-Phe-Ser-Gln-Arg-Tyr-Leu-Gly-Glu-Glu-Phe-Phe- Gly-Lys-Gly-Thr-Phe-Glu-Arg-Arg-Phe-Phe-Arg-Asp-Glu-Lys- Glu-Met-Gln-Asp-Tyr-Thr-Glu-Leu-Glu-Glu-Ala-Arg-Val-Glu- Ala-Ser-Leu-Ala-Asp-Asp-Gly-Thr-Val-Asp-Ser-Asp-Glu-Glu- Asp-Phe-Phe-Ser-Gly-Glu-Thr-Arg-Ser-Pro-Glu-Ala-Val-Tyr- Ser-Arg-Ile-Met-Met-Asn-Asn-Gly-Lys-Leu-Lys-Lys-Val-His- Ile-Arg-Arg-Tyr-Ile-Ala-Val-Ser-Ser-Asn-His-Gln-Ala-Arg- Pro-Asn-Ser-Phe-Ala-Glu-Phe-Leu-Asn-Lys-Val-Tyr-Ala-Asp- Gly-Ser-Met-Glu-Ser-Asp-Lys-Ile-Val-Phe-Lys-Val-Asn-Asn-Gln-Val- Val-Ser-Leu-Lys-Pro-Glu-Val-Ile-Ser-Asp-Gln- Tyr-Glu-Tyr- Lys-Tyr-Pro-Ala-Ile-Leu-Asp-Gly-Lys-Lys-Pro-Gly-Ile-Thr- Leu-Gly-Lys-Ala-Pro-Asp-Leu-Asn- Thr-Ala-Tyr-Lys-Ser-Ile- Leu-Ser-Gly-Met-Lys-Ala-Ala-Lys-Leu-Asp-Pro-Asp-Asp-Val- Cys-Ser-Tyr-Leu-Ala- Ala-Ala-Met-His-Leu-Phe-Glu-Gly-Val- Cys-Pro-Glu-Asp-Trp-Val-Ser-Tyr-Gly-Ile-Val-Ile-Ala-Lys- Lys-Gly- Glu-Lys-Ile-Asn-Pro-Ser-Val-Ile-Val-Asp-Ile-Val- Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp-Ala-Gln-Ala-Gly-Gly- Thr- Asp-Val-Ile-Arg-Asp-Pro-Thr-Met-Ala-Glu-His-Ala-Ser-Leu- Val-Gly-Leu-Leu-Leu-Cys-Leu-Tyr-Arg-Leu- Ser-Lys-Ile-Val- Gly-Gln-Asn-Thr-Ala-Asn-Tyr-Lys-Thr-Asn-Val-Ala-Asp-Arg- Met-Glu-Gln-Ile-Phe-Glu-Thr- Ala-Pro-Phe-Ala-Lys-Val-Val- Glu-His-His-Thr-Leu-Met-Thr-Thr-His-Lys-Met-Cys-Ala-Asn- Trp-Ser-Thr-Ile- Pro-Asn-Phe-Arg-Phe-Leu-Val-Gly-Thr-Tyr- Asp-Met-Phe-Phe-Ala-Arg-Val-Glu-His-Ile-Tyr-Ser-Ala-Leu- Arg- Val-Gly-Thr-Val-Val-Thr-Ala-Tyr-Glu-Asp-Cys-Ser-Gly- Leu-Val-Ser-Phe-Thr-Gly-Phe-Ile -Lys-Gln-Ile-Asn-Leu-Ser- Pro-Arg-Asp-Ala-Leu-Leu-Tyr-Phe-Phe-His-Lys-Asn-Phe-Glu- Gly-Glu-Ile-Lys-Arg -Met-Phe-Glu-Pro-Gly-Gln-Glu-Thr-Ala- Val-Pro-His-Ser-Tyr-Phe-Ile-His-Phe-Arg-Ala-Leu-Gly-Leu- Ser-Gly-Lys-Ser-Pro-Tyr-Ser-Ser-Asn-Ala-Val-Gly-His-Thr- Phe-Asn-Leu-Ile-His-Phe-Val-Gly-Cys-Tyr-Met- Gly-Gln-Ile- Arg-Ser-Leu-Asn-Ala-Thr-Val-Ile-Gln-Thr-Cys-Ala-Pro-Leu- Lys-Gly-Ala-Phe-Ser-Gln-Arg-Tyr- Leu-Gly-Glu-Glu-Phe-Phe- Gly-Lys-Gly-Thr-Phe-Glu-Arg-Arg-Phe-Phe-Arg-Asp-Glu-Lys- Glu-Met-Gln-Asp-Tyr- Thr-Glu-Leu-Glu-Glu-Ala-Arg-Val-Glu- Ala-Ser-Leu-Ala-Asp-Asp-Gly-Thr-Val-Asp-Ser-Asp-Glu-Glu- Asp-Phe- Phe-Ser-Gly-Glu-Thr-Arg-Ser-Pro-Glu-Ala-Val-Tyr- Ser-Arg-Ile-Met-Met-Asn-Asn-Gly-Lys-Leu-Lys-Lys-Val- His- Ile-Arg-Arg-Tyr-Ile-Ala-Val-Ser-Ser-Asn-His-Gln-Ala-Arg- Pro-Asn-Ser-Phe-Ala-Glu-Phe-Leu-Asn-Lys- Val-Tyr-Ala-Asp- Gly-Ser-
(II) (II)
Selon un autre mode de réalisation avantageux, ledit peptide est codé par le gène de la protéine M1 et présente une séquence en acides aminés qui répond à la formule III ci-après : Met-Ser-Lys-Asp-Leu-Val-His-Pro-Ser-Leu-Ile-Arg-Ala-Gly- Ile-Val-Glu-Leu-Glu-Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile- Asn-Arg-Thr-Ile-Glu-Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu- Pro-Leu-Tyr-Val-Asp-Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu- Arg-Ile-Glu-Asp-Lys-Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu- Glu-Arg-Asp-Glu-Gly-Ser-Ser-Glu-Glu-Asp-Asn-Tyr-Leu-Ser- Glu-Gly-Gln-Asp-Pro-Leu-Ile-Pro-Phe-Gln-Asn-Phe-Leu-Asp- Glu-Ile-Gly-Ala-Arg-Ala-Val-Lys-Arg-Leu-Lys-Thr-Gly-Glu- Gly-Phe-Phe-Arg-Val-Trp-Ser-Ala-Leu-Ser-Asp-Asp-Ile-Lys- Gly-Tyr-Val-Ser-Thr-Asn-Ile-Met-Thr-Ser-Gly-Glu-Arg-Asp- Thr-Lys-Ser-Ile-Gln-Ile-Gln-Thr-Glu-Pro-Thr-Ala-Ser-Val- Ser-Ser-Gly-Asn-Glu-Ser-Arg-His-Asp-Ser-Glu-Ser-Met-His- Asp-Pro-Asn-Asp-Lys-Lys-Asp-His-Thr-Pro-Asp-His-Asp-Val- Val-Pro-Asp-Ile-Glu-Ser-Ser-Thr-Asp-Lys-Gly-Glu-Ile-Arg- Asp-Ile-Glu-Gly-Glu-Val-Ala-His-Gln-Val-Ala-Glu-Ser-Phe- Ser-Lys-Lys-Tyr-Lys-Phe-Pro-Ser-Arg-Ser-Ser-Gly-Ile-Phe- Leu-Trp-Asn-Phe-Glu-Gln-Leu-Lys-Met-Asn-Leu-Asp-Asp-Ile- Val-Lys-Ala-Ala-Met-Asn-Val-Pro-Gly-Val-Glu-Arg-Ile-Ala- Glu-Lys-Gly-Gly-Lys-Leu-Pro-Leu-Arg-Cys-Ile-Leu-Gly-Phe- Val-Ala-Leu-Asp-Ser-Ser-Lys-Arg-Phe-Arg-Leu-Leu-Ala-Asp- Asn-Asp-Lys-Val-Ala-Arg-Leu-Ile-Gln-Glu-Asp-Ile-Asn-Ser- Tyr-Met-Ala-Arg-Leu-Glu-Glu-Ala-Glu- (IIl) According to another advantageous embodiment, said peptide is coded by the gene for the protein M1 and has an amino acid sequence which corresponds to formula III below: Met-Ser-Lys-Asp-Leu-Val-His- Pro-Ser-Leu-Ile-Arg-Ala-Gly- Ile-Val-Glu-Leu-Glu-Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile- Asn-Arg-Thr-Ile- Glu-Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu- Pro-Leu-Tyr-Val-Asp-Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu- Arg- Ile-Glu-Asp-Lys-Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu- Glu-Arg-Asp-Glu-Gly-Ser-Ser-Glu-Glu-Asp-Asn-Tyr- Leu-Ser- Glu-Gly-Gln-Asp-Pro-Leu-Ile-Pro-Phe-Gln-Asn-Phe-Leu-Asp- Glu-Ile-Gly-Ala-Arg-Ala-Val-Lys-Arg- Leu-Lys-Thr-Gly-Glu- Gly-Phe-Phe-Arg-Val-Trp-Ser-Ala-Leu-Ser-Asp-Asp-Ile-Lys- Gly-Tyr-Val-Ser-Thr-Asn- Ile-Met-Thr-Ser-Gly-Glu-Arg-Asp- Thr-Lys-Ser-Ile-Gln-Ile-Gln-Thr-Glu-Pro-Thr-Ala-Ser-Val- Ser-Ser-Gly- Asn-Glu-Ser-Arg-His-Asp-Ser-Glu-Ser-Met-His- Asp-Pro-Asn-Asp-Lys-Lys-Asp-His-Thr-Pro-Asp-His-Asp-Val- Val-Pro-Asp-Ile-Glu-Ser-Ser-Thr-Asp-Lys-Gly-Glu-Ile-Arg- Asp-Ile-Glu -Gly-Glu-Val-Ala-His-Gln-Val-Ala-Glu-Ser-Phe- Ser-Lys-Lys-Tyr-Lys-Phe-Pro-Ser-Arg-Ser-Ser-Gly-Ile-Phe - Leu-Trp-Asn-Phe-Glu-Gln-Leu-Lys-Met-Asn-Leu-Asp-Asp-Ile-Val-Lys-Ala-Ala-Met-Asn-Val-Pro-Gly-Val-Glu -Arg-Ile-Ala- Glu-Lys-Gly-Gly-Lys-Leu-Pro-Leu-Arg-Cys-Ile-Leu-Gly-Phe- Val-Ala-Leu-Asp-Ser-Ser-Lys-Arg -Phe-Arg-Leu-Leu-Ala-Asp- Asn-Asp-Lys-Val-Ala-Arg-Leu-Ile-Gln-Glu-Asp-Ile-Asn-Ser- Tyr-Met-Ala-Arg-Leu -Glu-Glu-Ala-Glu- (IIl)
Selon un autre mode de réalisation avantageux, ledit peptide est codé par le gène de la protéine M2 et présente une composition en acides aminés qui répond à la formule IV ci-après : According to another advantageous embodiment, said peptide is encoded by the protein M2 gene and has an amino acid composition which corresponds to formula IV below:
Met-Asn-Phe-Leu-Lys-Lys-Met-Ile-Lys-Ser-Cys-Lys-Asp-Glu- Glu-Thr-Gln-Lys-Tyr-Pro-Ser-Ala-Ser-Ala-Pro-Pro-Asp-Asp-
Asp-Asp-Ile-Trp-Met-Pro-Pro-Pro-Glu-Tyr-Val-Pro-Leu-Thr-Gln-Val-Lys-Gly-Lys-Ala-Ser-Val-Arg-Asn-Phe-Cys-Ile-Ser-Gly-Glu-Val-Lys-Ile-Cys-Ser-Pro-Asn-Gly-Tyr-Ser-Phe-Lys-Ile-Leu-Arg-His-Ile-Leu-Lys-Ser-Phe-Asp-Asn-Val-Tyr-Ser-Gly-Asn-Arg-Arg-Met -Ile-Gly-Leu-Val-Lys-Val-Val-Ile-Gly-Leu-Val-Leu-Ser-Gly -Ser-Pro -Val-Pro-Glu-Gly-Met-Asn-Trp-Val-Tyr-Lys-Leu-Arg-Arg-Thr-Leu-Ile-Phe-Gln-Trp-Ala-Glu-Ser-His-Gly-Pro-Leu-Glu-Gly-Glu-Glu-Leu-Glu-Tyr-Ser-Gln-Glu-Ile-Thr-Trp-Asp-Asp-Glu-Ala-Glu-Phe-Val-Gly-Leu-Gln-Ile-Arg-Val-Ser-Ala-Arg-Gln-Cys-His-Ile-Gln-Gly-Arg-Leu-Trp-Cys-Ile -Asn-Met-Asn-Ser-Arg-Ala-Cys-Gln-Leu-Trp-Ala-Asp-Met-Ile-Leu-Gln-Thr-Gln-Gln-Ser-Pro-Asp--Asp-Glu-Asn-Thr-Ser-Leu-Leu-Leu-Glu-Met-Asn-Phe-Leu-Lys-Lys-Met-Ile-Lys-Ser-Cys-Lys-Asp-Glu-Thr-Gln-Lys-Tyr-Pro-Ser-Ala-Ser-Ala-Pro- Pro-Asp-Asp- Asp-Asp-Ile-Trp-Met-Pro-Pro-Pro-Glu-Tyr-Val-Pro-Leu-Thr-Gln-Val-Lys-Gly-Lys-Ala-Ser-Val-Arg-Asn-Phe- Cys-Ile-Ser-Gly-Glu-Val-Lys-Ile-Cys-Ser-Pro-Asn-Gly-Tyr-Ser-Phe-Lys-Ile-Leu-Arg-His-Ile-Leu-Lys-Ser- Phe-Asp-Asn-Val-Tyr-Ser-Gly-Asn-Arg-Arg-Met -Ile-Gly-Leu-Val-Lys-Val-Val-Ile-Gly-Leu-Val-Leu-Ser-Gly - Ser-Pro -Val-Pro-Glu-Gly-Met-Asn-Trp-Val-Tyr-Lys-Leu-Arg-Arg-Thr-Leu-Ile-Phe-Gln-Trp-Ala-Glu-Ser-His- Gly-Pro-Leu-Glu-Gly-Glu-Glu-Leu-Glu-Tyr-Ser-Gln-Glu-Ile-Thr-Trp-Asp-Asp-Glu-Ala-Glu-Phe-Val-Gly-Leu- Gln-Ile-Arg-Val-Ser-Ala-Arg-Gln-Cys-His-Ile-Gln-Gly-Arg-Leu-Trp-Cys-Ile -Asn-Met-Asn-Ser-Arg-Ala-Cys- Gln-Leu-Trp-Ala-Asp-Met-Ile-Leu-Gln-Thr-Gln-Gln-Ser-Pro-Asp - Asp-Glu-Asn-Thr-Ser-Leu-Leu-Leu-Glu-
(IV) (IV)
Selon un mode de réalisation avantageux ledit peptide est codé par le gène de la glycoprotéine G et est caractérisé par une séquence en amino acides qui répond à la formule V ci-après : According to an advantageous embodiment, said peptide is coded by the glycoprotein G gene and is characterized by an amino acid sequence which corresponds to formula V below:
Met- Asn- Ile- Pro- Cys- Phe- Val- Val-Ile-Leu- Ser- Leu- Ala- Thr-Met- Asn- Ile- Pro- Cys- Phe- Val- Val-Ile-Leu- Ser- Leu- Ala- Thr-
Thr-His-Ser-Leu-Gly Glu-Phe-Pro-Leu-Tyr-Thr-Ile-Pro-Glu- Lys-Ile-Glu-Lys-Trp-Thr Pro-Ile-Asp-Met-Ile-His-Leu-Ser- Cys-Pro-Asn-Asn-Leu-Leu Ser-Glu-Glu Glu-Gly-Cys-Asn-Ala- Glu-Ser-Ser-Phe-Thr-Tyr Phe-Glu-Leu Lys-Ser Gly Tyr-Leu- Ala-His-Gln-Lys-Val-Pro Gly-Phe-Thr-Cys-Thr-Gly Val-Val- Asn-Glu-Ala-Glu-Thr-Tyr-Thr-Asn-Phe-Val-Gly--Tyr Val-Thr- Thr-Thr-Phe-Lys-Arg-Lys-His Phe-Arg-Pro-Thr-Val-Ala-Ala- Cys-Arg-Asp-Ala-Tyr-Asn-Trp-Lys Val Ser-Gly-Asp Pro-Arg- Tyr Glu-Glu-Ser-Leu-His-Thr-Pro-Tyr-Pro-Asp-Ser-Ser-Trp- Leu-Arg-Thr-Val-Thr-Thr-Thr-Lys-Glu-Ser-Leu-Leu-Ile-Ile- Ser-Pro-Ser-Ile-Val-Glu-Met-Asp-Ile-Tyr-Gly-Arg-Thr-Leu- His-Ser-Pro-Met-Phe-Pro-Ser-Gly-Val-Cys-Ser-Asn-Val-Tyr- Pro-Ser-Val-Pro-Ser-Cys-Glu-Thr-Asn-His-Asp-Tyr-Thr-Leu- Trp-Leu-Pro-Glu-Asp-Pro-Ser-Leu-Ser-Leu-Val-Cys-Asp-Ile- Phe-Thr-Ser-Ser-Asn-Gly-Lys-Lys-Ala-Met-Asn-Gly-Ser-Arg- Ile-Cys-Gly-Phe-Lys-Asp-Glu-Arg-Gly-Phe-Tyr-Arg-Ser-Leu- Lys-Gly-Ala-Cys-Lys-Leu-Thr-Leu-Cys-Gly-Arg-Pro-Gly-Ile- Arg-Leu-Phe-Asp-Gly-Thr-Trp-Val-Ser-Phe-Thr-Lys-Pro-Asp- Val-His-Val-Trp-Cys-Thr-Pro-Asn-Gln-Leu-Ile-Asn-Ile-His- Asn-Asp-Arg-Leu-Asp-Glu-Ile-Glu-His-Leu-Ile-Val-Glu-Asp- Ile-Ile-Lys-Lys-Arg-Glu-Glu-Cys-Leu-Asp-Thr-Leu-Glu-Thr- Ile-Leu-Met-Ser-Gln-Ser-Val-Ser-Phe-Arg-Arg-Leu-Ser-His- Phe-Arg-Lys-Leu-Val-Pro-Gly-Tyr-Gly-Lys-Ala-Tyr-Thr-Ile- Leu-Asn-Gly-Ser-Leu-Met-Glu-Thr-Asn-Val-Tyr-Tyr-Lys-Arg- Val-Asp-Lys-Trp-Ala-Asp-Ile-Leu-Pro-Ser-Lys-Gly-Cys-Leu- Lys-Val-Gly-Gln-Gln-Cys-Met-Glu-Pro-Val-Lys-Gly-Val-Leu- Phe-Asn-Gly-Ile-Ile-Lys-Gly-Pro-Asp-Gly-Gin-Ile-Leu-Ile- Pro-Glu-Met-Gln-Ser-Glu-Gln-Leu-Lys-Gln-His-Met-Asp-Leu- Leu-Lys-Ala-Ala-Val-Phe-Pro-Leu-Arg-His-Pro-Leu-Ile-Ser- Arg-Glu-Ala-Val-Phe-Lys-Lys-Asp-Gly-Asp-Ala Asp-Asp Phe- Val-Asp-Leu-His-Met-Pro-Asp-Val-His-Lys-Ser-Val-Ser Asp-
Val-Asp-Leu-Gly-Leu-Pro-His-Trp-Gly-Phe-Trp-Met-Leu-Ile-Thr-His-Ser-Leu-Gly Glu-Phe-Pro-Leu-Tyr-Thr-Ile-Pro-Glu- Lys-Ile-Glu-Lys-Trp-Thr Pro-Ile-Asp-Met-Ile-His- Leu-Ser- Cys-Pro-Asn-Asn-Leu-Leu Ser-Glu-Glu Glu-Gly-Cys-Asn-Ala- Glu-Ser-Ser-Phe-Thr-Tyr Phe-Glu-Leu Lys-Ser Gly Tyr-Leu- Ala-His-Gln-Lys-Val-Pro Gly-Phe-Thr-Cys-Thr-Gly Val-Val- Asn-Glu-Ala-Glu-Thr-Tyr-Thr-Asn-Phe-Val- Gly - Tyr Val-Thr- Thr-Thr-Phe-Lys-Arg-Lys-His Phe-Arg-Pro-Thr-Val-Ala-Ala- Cys-Arg-Asp-Ala-Tyr-Asn-Trp-Lys Val Ser-Gly-Asp Pro-Arg- Tyr Glu-Glu-Ser-Leu-His-Thr-Pro-Tyr-Pro-Asp-Ser-Ser-Trp- Leu-Arg-Thr-Val-Thr-Thr-Thr -Lys-Glu-Ser-Leu-Leu-Ile-Ile- Ser-Pro-Ser-Ile-Val-Glu-Met-Asp-Ile-Tyr-Gly-Arg-Thr-Leu- His-Ser-Pro-Met -Phe-Pro-Ser-Gly-Val-Cys-Ser-Asn-Val-Tyr- Pro-Ser-Val-Pro-Ser-Cys-Glu-Thr-Asn-His-Asp-Tyr-Thr-Leu- Trp -Leu-Pro-Glu-Asp-Pro-Ser-Leu-Ser-Leu-Val-Cys-Asp-Ile- Phe-Thr-Ser-Ser-Asn-Gly-Lys-Lys-Ala-Met-Asn-Gly -Ser-Arg- Ile-Cys-Gly-Phe-Lys-Asp-Glu-Arg-Gly-Phe-Tyr-Arg-Ser-Leu- Lys-Gly-Ala-Cys-Lys-Leu-Thr-Leu-Cys -Gly-Arg-Pro-Gly-Ile- Arg-Leu-Phe-Asp-Gly-Thr-Trp-Va l-Ser-Phe-Thr-Lys-Pro-Asp- Val-His-Val-Trp-Cys-Thr-Pro-Asn-Gln-Leu-Ile-Asn-Ile-His- Asn-Asp-Arg-Leu- Asp-Glu-Ile-Glu-His-Leu-Ile-Val-Glu-Asp- Ile-Ile-Lys-Lys-Arg-Glu-Glu-Cys-Leu-Asp-Thr-Leu-Glu-Thr- Ile- Leu-Met-Ser-Gln-Ser-Val-Ser-Phe-Arg-Arg-Leu-Ser-His- Phe-Arg-Lys-Leu-Val-Pro-Gly-Tyr-Gly-Lys-Ala-Tyr- Thr-Ile- Leu-Asn-Gly-Ser-Leu-Met-Glu-Thr-Asn-Val-Tyr-Tyr-Lys-Arg- Val-Asp-Lys-Trp-Ala-Asp-Ile-Leu-Pro- Ser-Lys-Gly-Cys-Leu- Lys-Val-Gly-Gln-Gln-Cys-Met-Glu-Pro-Val-Lys-Gly-Val-Leu- Phe-Asn-Gly-Ile-Ile-Lys- Gly-Pro-Asp-Gly-Gin-Ile-Leu-Ile- Pro-Glu-Met-Gln-Ser-Glu-Gln-Leu-Lys-Gln-His-Met-Asp-Leu- Leu-Lys-Ala- Ala-Val-Phe-Pro-Leu-Arg-His-Pro-Leu-Ile-Ser- Arg-Glu-Ala-Val-Phe-Lys-Lys-Asp-Gly-Asp-Ala Asp-Asp Phe- Val- Asp-Leu-His-Met-Pro-Asp-Val-His-Lys-Ser-Val-Ser Asp- Val-Asp-Leu-Gly-Leu-Pro-His-Trp-Gly-Phe-Trp-Met-Leu-Ile-
Gly-Ala-Thr-Ile-Val-Ala-Phe-Val-Val-Leu-Val-Cys-Leu-Leu-Gly-Ala-Thr-Ile-Val-Ala-Phe-Val-Val-Leu-Val-Cys-Leu-Leu-
Arg-Val-Cys-Cys-Lys-Arg-Val-Arg-Arg-Arg-Arg-Ser-Gly-Arg-Arg-Val-Cys-Cys-Lys-Arg-Val-Arg-Arg-Arg-Arg-Ser-Gly-Arg-
Ala-Thr-Gln-Glu-Ile-Pro-Leu-Ser-Phe-Pro-Ser-Ala-Pro-Val-Ala-Thr-Gln-Glu-Ile-Pro-Leu-Ser-Phe-Pro-Ser-Ala-Pro-Val-
Pro-Arg-Ala-Lys- Val-Val-Ser-Ser-Trp-Glu-Ser-Tyr-Lys-Pro-Arg-Ala-Lys- Val-Val-Ser-Ser-Trp-Glu-Ser-Tyr-Lys-
Gly-Leu-Pro-Gly-Thr-Gly-Leu-Pro-Gly-Thr-
(V) (V)
Selon un autre mode de réalisation avantageux, ledit peptide est codé par le gène de la protéine L et présente l'une des séquences en acides aminés suivante : According to another advantageous embodiment, said peptide is coded by the L protein gene and has one of the following amino acid sequences:
- la séquence de formule VI ci-après : - the sequence of formula VI below:
Met-Met-Asp-Val-Thr-Glu-Val-Tyr-Asp-Asp-Pro-Ile-Asp-Pro- Val-Glu-Pro-Glu-Gly-Glu-Trp-Asn-Ser-Ser-Pro-Val-Val-Pro- - - - - - - - - - - - - - - - - - - - - - M Met-Ile-Gln-Trp-Leu-Thr-Ser-Gly-Asn-Arg-Pro- Ser-Arg-Met-...-Val-Thr-Glu-Asn-Thr-Thr-Arg-Ser-Tyr-Lys- Val-Leu-Arg-Ala-Leu-Phe-Lys-Gly-Val-Asp-Ile-Ala-Thr-Ile- Lys-Ile-Gly-Gly-Val-Gly-Ala-Gln-Ala-Met-Met-Gly-Leu-Trp- Val-Leu-Gly-Ser-His-Ser-Glu-Ser-Ser-Arg-Ser-Arg-Lys-Cys- Leu-Ala-Asp-Leu-Ser-Ala-Phe-Tyr-Gln-Arg-Thr-Leu-Pro-Ile- Glu-Ser-Ile-Leu-Asn-Gln-His-Leu-Asn-Glu-Gln-Arg-Thr-Thr- Asp-Pro-Arg-Glu-Gly-Val-Leu-Ser-Gly-Leu-Asn-Arg-Val-Ser- Tyr-Asp-Gln-Ser-Phe-Gly-Arg-Tyr-Leu-Gly-Asn-Leu-Tyr-Ser- Ser-Tyr-Leu-Leu-Phe-His-Val-Ile-Ile-Leu-Tyr-Met-Asn-Ala- Leu-Asp-Trp-Glu-Glu- - -Thr-Ile-Leu-Ala-Leu-Trp-Arg- Asp-Ile-Thr-Ser-Ile-Asp-Ile-Lys-Asn-Asp-Arg-Val-Tyr-Phe- Lys-Asp-Pro-Leu-Trp-Gly-Lys-Leu-Leu-Val-Thr-Lys-Asp-Phe- Val-Tyr-Ala-His-Asn-Ser-Asn-Cys-Leu-Phe-Asp-Lys-Asn-Tyr- Thr-Leu-Met-Leu-Lys-Asp-Leu-Phe-Arg-Ala-Arg-Phe-Asn-Ser- Leu-Leu-Ile-Leu-Val-Ser-Pro-Pro-Asp-Ser-Arg-Tyr-Ser-Asp- Asp-Leu-Ala-Ala-Asn-Leu-Cys-Arg-Leu-Tyr-Ile-Ser-Gly-Asp- Arg-Leu-Leu-Ser-Ser-Cys-Gly-Asn-Ala-Gly-Tyr-Asp-Val-Ile- Lys-Met-Leu-Glu-Pro-Cys-Val-Val-Asp-Leu-Leu-Val-Gln-Arg- Ala-Glu-Thr-Phe-Arg-Pro-Leu-Ile-His-Ser-Leu-Gly-Glu-Phe- Pro-Ala-Phe-Ile-Lys-Asp-Lys-Thr-Thr-Gln-Leu-Ile-Gly- Phe-Gly-Pro-Cys-Asp-Tyr-Asn-Phe-Phe-Ser-Met-Leu-Gln-Asn- Phe-Asp-Asn-Ile-His-Asp-Leu-Val-Phe-Ile-Tyr-Gly-Cys-Tyr- Arg-His-Trp-Gly-His-Pro-Tyr-Ile-Asp-Tyr-Arg-Lys-Gly-Leu- Ser-Lys-Leu-Phe-Asp-Gln-Val-His-Met-Lys-Lys-Thr-Ile-Asp- Gln-Gln-Tyr-Gln-Glu-Arg-Leu-Ala-Ser-Asp-Leu-Ala-Arg-Lys- Ile-Leu-Arg-Trp-Gly-Phe-Glu-Lys-Tyr-Ser-Lys-Trp-Tyr-Leu- Asp-Thr-Gly-Val-Ile-Pro-Lys-Asp-His-Pro-Leu-Ala-Pro-Tyr- Ile-Ala-Thr-Gln-Thr-Trp-Pro-Pro-Lys-His-Val-Val-Asp-Leu- Leu-Gly-Asp-Ser-Trp-His-Thr-Leu-Pro-Met-Thr-Met-Met-Asp-Val-Thr-Glu-Val-Tyr-Asp-Asp-Pro-Ile-Asp-Pro- Val-Glu-Pro-Glu-Gly-Glu-Trp-Asn-Ser-Ser-Pro- Val-Val-Pro- - - - - - - - - - - - - - - - - - - - - - M Met-Ile-Gln-Trp-Leu-Thr-Ser-Gly-Asn-Arg-Pro- Ser-Arg-Met -...- Val-Thr-Glu-Asn-Thr-Thr-Arg-Ser-Tyr-Lys- Val-Leu-Arg-Ala-Leu-Phe-Lys-Gly-Val-Asp- Ile-Ala-Thr-Ile- Lys-Ile-Gly-Gly-Val-Gly-Ala-Gln-Ala-Met-Met-Gly-Leu-Trp- Val-Leu-Gly-Ser-His-Ser-Glu- Ser-Ser-Arg-Ser-Arg-Lys-Cys- Leu-Ala-Asp-Leu-Ser-Ala-Phe-Tyr-Gln-Arg-Thr-Leu-Pro-Ile- Glu-Ser-Ile-Leu- Asn-Gln-His-Leu-Asn-Glu-Gln-Arg-Thr-Thr- Asp-Pro-Arg-Glu-Gly-Val-Leu-Ser-Gly-Leu-Asn-Arg-Val-Ser- Tyr- Asp-Gln-Ser-Phe-Gly-Arg-Tyr-Leu-Gly-Asn-Leu-Tyr-Ser- Ser-Tyr-Leu-Leu-Phe-His-Val-Ile-Ile-Leu-Tyr-Met- Asn-Ala- Leu-Asp-Trp-Glu-Glu- - -Thr-Ile-Leu-Ala-Leu-Trp-Arg- Asp-Ile-Thr-Ser-Ile-Asp-Ile-Lys-Asn-Asp- Arg-Val-Tyr-Phe- Lys-Asp-Pro-Leu-Trp-Gly-Lys-Leu-Leu-Val-Thr-Lys-Asp-Phe- Val-Tyr-Ala-His-Asn-Ser-Asn- Cys-Leu-Phe-Asp-Lys-Asn-Tyr- Thr-Leu-Met-Leu-Lys-Asp-Leu-Phe-Arg-Ala-Arg-Phe-Asn-Ser- Leu -Leu-Ile-Leu-Val-Ser-Pro-Pro-Asp-Ser-Arg-Tyr-Ser-Asp- Asp-Leu-Ala-Ala-Asn-Leu-Cys-Arg-Leu-Tyr-Ile-Ser -Gly-Asp- Arg-Leu-Leu-Ser-Ser-Cys-Gly-Asn-Ala-Gly-Tyr-Asp-Val-Ile- Lys-Met-Leu-Glu-Pro-Cys-Val-Val-Asp -Leu-Leu-Val-Gln-Arg- Ala-Glu-Thr-Phe-Arg-Pro-Leu-Ile-His-Ser-Leu-Gly-Glu-Phe- Pro-Ala-Phe-Ile-Lys-Asp -Lys-Thr-Thr-Gln-Leu-Ile-Gly- Phe-Gly-Pro-Cys-Asp-Tyr-Asn-Phe-Phe-Ser-Met-Leu-Gln-Asn- Phe-Asp-Asn-Ile -His-Asp-Leu-Val-Phe-Ile-Tyr-Gly-Cys-Tyr- Arg-His-Trp-Gly-His-Pro-Tyr-Ile-Asp-Tyr-Arg-Lys-Gly-Leu- Ser -Lys-Leu-Phe-Asp-Gln-Val-His-Met-Lys-Lys-Thr-Ile-Asp- Gln-Gln-Tyr-Gln-Glu-Arg-Leu-Ala-Ser-Asp-Leu-Ala -Arg-Lys- Ile-Leu-Arg-Trp-Gly-Phe-Glu-Lys-Tyr-Ser-Lys-Trp-Tyr-Leu- Asp-Thr-Gly-Val-Ile-Pro-Lys-Asp-His -Pro-Leu-Ala-Pro-Tyr- Ile-Ala-Thr-Gln-Thr-Trp-Pro-Pro-Lys-His-Val-Val-Asp-Leu- Leu-Gly-Asp-Ser-Trp-His -Thr-Leu-Pro-Met-Thr-
(VI) (VI)
- la séquence de formule VII ci-après : - the sequence of formula VII below:
Glu-Ser-Phe-Leu-Asn-Ser-Glu-Ile-His-Gly-Ile-Asn-Arg-Val-
Thr-Gln-Thr-Pro-Gln-Arg-Leu Glu-Ser-Phe-Leu-Asn-Ser-Glu-Ile-His-Gly-Ile-Asn-Arg-Val- Thr-Gln-Thr-Pro-Gln-Arg-Leu
(VII) (VII)
- la séquence de formule VIII ci-après : - the sequence of formula VIII below:
Val-Asp-Leu-Gly-Pro- Lys-Ser-Ser-Val-Ala-Cys-Gly-Cys-Tyr-Thr-Arg-Glu-Val-Gly- Asn-Pro-Arg-Ile-Ser-Val-Ser-Val-Leu-Pro-Ser-Phe-Asp-Pro- Ser-Phe-Leu-Ser-Arg-Gly-Pro-Leu-Lys-Gly-Tyr-Leu-Gly-Ser- Ser-Thr-Ser-Met-Ser-Thr-Gln-Leu-Phe-His-Ser-Trp-Glu-Lys- Val-Thr-Asn-Val-His-Val-Val-Lys-Arg-Ala-Leu-Ser-Leu-Lys- Glu-Ser-Ile-Asn-Trp-Phe-Val-Ser-Arg-Glu-Ser-Asn-Leu-Ala- Lys-Thr-Leu-Ile-Gly-Asn-Ile-Leu-Ser-Leu-Thr-Gly-Pro-Ile- Phe-Ser-Ile-Glu-Glu-Ala-Pro-Val-Phe-Lys-Arg-Thr-Gly-Ser- Ala-Leu-His-Arg-Phe-Lys-Ser-Ala-Arg-Tyr-Ser-Glu-Gly-Gly- Tyr-Pro-Ala-Val-Cys-Pro Val-Asp-Leu-Gly-Pro- Lys-Ser-Ser-Val-Ala-Cys-Gly-Cys-Tyr-Thr-Arg-Glu-Val-Gly- Asn-Pro-Arg-Ile-Ser-Val- Ser-Val-Leu-Pro-Ser-Phe-Asp-Pro- Ser-Phe-Leu-Ser-Arg-Gly-Pro-Leu-Lys-Gly-Tyr-Leu-Gly-Ser- Ser-Thr-Ser- Met-Ser-Thr-Gln-Leu-Phe-His-Ser-Trp-Glu-Lys- Val-Thr-Asn-Val-His-Val-Val-Lys-Arg-Ala-Leu-Ser-Leu-Lys- Glu-Ser-Ile-Asn-Trp-Phe-Val-Ser-Arg-Glu-Ser-Asn-Leu-Ala- Lys-Thr-Leu-Ile-Gly-Asn-Ile-Leu-Ser-Leu-Thr- Gly-Pro-Ile- Phe-Ser-Ile-Glu-Glu-Ala-Pro-Val-Phe-Lys-Arg-Thr-Gly-Ser- Ala-Leu-His-Arg-Phe-Lys-Ser-Ala- Arg-Tyr-Ser-Glu-Gly-Gly- Tyr-Pro-Ala-Val-Cys-Pro
(VIII) (VIII)
- et la séquence de formule IX ci-après- and the sequence of formula IX below
Leu-Tyr-Asn-Ser-Pro-Val-Thr-Tyr-Tyr-Phe-Gly-Lys- Gln-Thr-Ile-Lys-Gly-Arg-Arg-Tyr-Leu-Ser-Trp-Ser-Trp-Ala- Asn-Ser-Ser-Pro-Ile-Phe-Lys-Lys-Val-Ala-Cys-Asn-Ser-Ser- Ile-Ser-Leu-Ser-Ser-His-Trp-Ile-Arg-Leu-Ile-Tyr-Lys-Ile- Val-Lys-Thr-Thr-Arg-Leu-Asn-Cys-Ser-Pro-Arg-Asp-Met-Leu- Arg-Glu-Thr-Glu-Ala-Cys-Leu-Arg-Thr-Tyr-Asn-Lys-Trp-Ile- Asn-Ile-Arg-Asp-Thr-Arg-Ser-Arg-Thr-Ser-Ile-Leu-Asp-Tyr- Cys-Cys-Leu Leu-Tyr-Asn-Ser-Pro-Val-Thr-Tyr-Tyr-Phe-Gly-Lys- Gln-Thr-Ile-Lys-Gly-Arg-Arg-Tyr-Leu-Ser-Trp-Ser-Trp- Ala- Asn-Ser-Ser-Pro-Ile-Phe-Lys-Lys-Val-Ala-Cys-Asn-Ser-Ser- Ile-Ser-Leu-Ser-Ser-His-Trp-Ile-Arg-Leu- Ile-Tyr-Lys-Ile- Val-Lys-Thr-Thr-Arg-Leu-Asn-Cys-Ser-Pro-Arg-Asp-Met-Leu- Arg-Glu-Thr-Glu-Ala-Cys-Leu- Arg-Thr-Tyr-Asn-Lys-Trp-Ile- Asn-Ile-Arg-Asp-Thr-Arg-Ser-Arg-Thr-Ser-Ile-Leu-Asp-Tyr- Cys-Cys-Leu
(IX) (IX)
Selon un mode de réalisation avantageux des peptides et/ou fragments peptidiques conformes à l'invention, ceux-ci sont obtenus par synthèse. According to an advantageous embodiment of the peptides and / or peptide fragments in accordance with the invention, these are obtained by synthesis.
La présente invention a également pour objet un vecteur, caractérisé en ce qu'il contient au moins une séquence nucleotidique ou une portion de cette séquence telle que définie ci-dessus. The present invention also relates to a vector, characterized in that it contains at least one nucleotide sequence or a portion of this sequence as defined above.
La présente invention a également pour objet un vecteur d'expression d'un peptide ou d'un fragment de peptide ou d'une combinaison de fragments de peptides du virus Mokola, caractérisé en ce qu'il est obtenu par recombinaison homologue entre un baculovirus de souche sauvage et un vecteur navette approprié comprenant au
moins : The present invention also relates to a vector for the expression of a peptide or of a peptide fragment or of a combination of fragments of peptides of the Mokola virus, characterized in that it is obtained by homologous recombination between a baculovirus. of wild strain and an appropriate shuttle vector comprising at least minus:
(1) des séquences régulatrices de l'expression du baculovirus ; (1) sequences regulating the expression of baculovirus;
(2) un polylinker approprié à l'insertion d'un gène ou d'au moins un fragment de gène du virus Mokola. (2) a polylinker suitable for the insertion of a gene or at least one gene fragment of the Mokola virus.
Selon un mode de réalisation avantageux du vecteur d'expression conforme à l'invention, ledit vecteur navette comprend : According to an advantageous embodiment of the expression vector in accordance with the invention, said shuttle vector comprises:
(1) un fragment de génome d'un baculovirus, comportant la région de contrôle en 5' du gène de la polyédrine ; (1) a genome fragment of a baculovirus, comprising the 5 'control region of the polyhedrin gene;
(2) un polylinker approprié à l'insertion d'un gène ou d'au moins un fragment de gène du virus Mokola ; (2) a polylinker suitable for the insertion of a gene or at least one gene fragment of the Mokola virus;
(3) les séquences de contrôle en 3' avec notamment le site de polyadénylation de la polyédrine, ledit vecteur navette permettant la multiplication de la construction. (3) the 3 ′ control sequences with in particular the polyadenylation site of the polyhedrin, said shuttle vector allowing the multiplication of the construction.
Selon une disposition avantageuse de ce mode de réalisation, le gène ou un fragment du gène de la glycoprotéine G du virus Mokola est inséré au niveau du polylinker, de manière à obtenir un vecteur d'expression dit chargé de ladite glycoprotéine, sous contrôle du promoteur du gène de la polyédrine. According to an advantageous arrangement of this embodiment, the gene or a fragment of the gene for the glycoprotein G of the Mokola virus is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said glycoprotein, under the control of the promoter of the polyhedrin gene.
Conformément à l'invention, ledit vecteur d'expression a été déposé sous le numéro 1-851 en date du 22 mars 1989 auprès de la Collection Nationale de Cultures de micro-organismes tenue par l'Institut Pasteur. In accordance with the invention, said expression vector was deposited under the number 1-851 dated March 22, 1989 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
Selon une autre disposition avantageuse de ce mode de réalisation, le gène ou un fragment du gène de la nucléoprotéine N est inséré au niveau du polylinker, de manière à obtenir un vecteur d'expression dit chargé de ladite nucléoprotéine, sous contrôle du promoteur du gène de la polyédrine. According to another advantageous arrangement of this embodiment, the gene or a fragment of the nucleoprotein N gene is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said nucleoprotein, under the control of the gene promoter polyhedrin.
Le processus d'expression d'au moins un peptide, un fragment de peptide ou une combinaison de frag
ments de peptides du virus Mokola, met en oeuvre un vecteur d'expression conforme à l'invention, dans des cellules de lépidoptère appropriées, notamment la chenille Spodoptera frugiperda, Sf9 en présence de baculovirus de type sauvage, pour l'obtention de baculovirus recombinants exprimant le polypeptide désiré. The process of expression of at least one peptide, a fragment of a peptide or a combination of frag peptides of the Mokola virus, implements an expression vector in accordance with the invention, in appropriate lepidopteran cells, in particular the caterpillar Spodoptera frugiperda, Sf9 in the presence of wild-type baculovirus, for obtaining recombinant baculoviruses expressing the desired polypeptide.
La sélection des baculovirus recombinants se fera sur la morphologie des plages obtenues. En effet, la présence de la polyédrine donne aux plages de virus sauvage un aspect réfringeant en microscopie optique lorsqu'on les éclaire par épiluminescence. A l'inverse, l'aspect terne des plages de virus recombinants, dû à l'absence de cristal, permet de les distinguer facilement. The selection of recombinant baculoviruses will be based on the morphology of the ranges obtained. Indeed, the presence of polyhedrin gives the beaches of wild virus a refractive aspect in light microscopy when they are illuminated by epiluminescence. Conversely, the dull appearance of the recombinant virus ranges, due to the absence of crystal, makes it easy to distinguish them.
Les avantages présentés par ce système d'expression sont multiples : The advantages presented by this expression system are multiple:
- les baculovirus sont aisément capables de glycosyler, phophoryler, palmityler, etc... Ceci permet d'envisager l'expression de nombreuses protéines ayant des modifications post-traductionnelles comme c'est le cas pour la glycoprotéine G ; - Baculoviruses are easily capable of glycosylating, phophoryling, palmityling, etc. This allows considering the expression of numerous proteins having post-translational modifications as is the case for glycoprotein G;
- le taux d'expression peut être extrêmement élevé, de l'ordre de 1 à 10 mg par litre de culture ; - the expression level can be extremely high, of the order of 1 to 10 mg per liter of culture;
- la culture en suspension des cellules de lé- pidoptères est possible ; - suspension culture of lepidopteran cells is possible;
- le fait que la polyédrine soit un gène tardif s'exprimant après que la réplication virale et la formation des nucléocapsides aient eu lieu, permet même d'exprimer des protéines toxiques pour la multiplication du virus. - the fact that polyhedrin is a late gene expressing itself after viral replication and the formation of nucleocapsides have taken place, even allows proteins toxic for the multiplication of the virus to be expressed.
La présente invention a également pour objet un vaccin contre le virus Mokola, à usage humain et/ou vétérinaire, caractérisé en ce qu'il comprend au moins un peptide et/ou un fragment de peptide conforme à l'invention, éventuellement associé à au moins un véhicule pharmaceutiquement acceptable.
Selon un mode de réalisation avantageux dudit vaccin, il comprend la glycoprotéine G et/ou un fragment de celle-ci et/ou la nucléoprotéine N ou un fragment de celle-ci. The present invention also relates to a vaccine against the Mokola virus, for human and / or veterinary use, characterized in that it comprises at least one peptide and / or a peptide fragment in accordance with the invention, optionally associated with at least one pharmaceutically acceptable vehicle. According to an advantageous embodiment of said vaccine, it comprises the glycoprotein G and / or a fragment thereof and / or the nucleoprotein N or a fragment thereof.
L'association de ces deux peptides viraux présente les avantages suivants : ils interviennent à des niveaux différents de la réponse immune ; en effet la glycoprotéine G induit préférentiellement la formation d'anticorps neutralisants alors que la nucléoprotéine N intervient surtout dans l'immunité à médiation cellulaire. The combination of these two viral peptides has the following advantages: they intervene at different levels of the immune response; in fact the glycoprotein G preferentially induces the formation of neutralizing antibodies whereas the nucleoprotein N intervenes especially in cell-mediated immunity.
La présente invention a également pour objet un vaccin polyvalent des Lyssavirus, caractérisé en ce qu'il comprend au moins un peptide et/ou fragment peptidique conforme à l'invention, éventuellement associé à au moins un peptide et/ou un fragment peptidique d'.au moins un autre serotype de Lyssavirus. The present invention also relates to a polyvalent Lyssavirus vaccine, characterized in that it comprises at least one peptide and / or peptide fragment in accordance with the invention, optionally associated with at least one peptide and / or a peptide fragment of at least one other Lyssavirus serotype.
On peut citer notamment les Lyssavirus de serotype 1 (virus rabique), les Lyssavirus de serotype 2 (virus Lagos bat), les Lyssavirus de serotype 4 (virus Duvenhage), et les Lyssavirus isolés sur les chauves-souris d'Europe, proches du serotype 4. Mention may in particular be made of the Lyssaviruses of serotype 1 (rabies virus), the Lyssaviruses of serotype 2 (Lagos bat virus), the Lyssaviruses of serotype 4 (Duvenhage virus), and the Lyssaviruses isolated on European bats, close to serotype 4.
Conformément à l'invention lesdits peptides et/ou fragments peptidiques sont avantageusement associés à un support approprié et/ou un adjuvant acceptable, notamment les adjuvants classiques des vaccins humains et vétérinaires. In accordance with the invention, said peptides and / or peptide fragments are advantageously associated with an appropriate support and / or an acceptable adjuvant, in particular the conventional adjuvants of human and veterinary vaccines.
La présente invention a également pour objet des anticorps monoclonaux spécifiques des peptides et/ou fragments peptidiques du virus Mokola, caractérisés en ce qu'ils résultent de l'immunisation de mammifères, notamment de rongeurs, et plus particulièrement de souris, par des peptides et/ou fragments peptidiques conformes à l'invention. The present invention also relates to monoclonal antibodies specific for the peptides and / or peptide fragments of the Mokola virus, characterized in that they result from the immunization of mammals, in particular rodents, and more particularly of mice, by peptides and / or peptide fragments in accordance with the invention.
La présente invention a également pour objet un procédé immunologique de détection d'anticorps anti
peptides et/ou fragments peptidiques du virus Mokola, qui consiste à détecter les anticorps anti-Mokola éventuellement présents dans un échantillon biologique à l'aide d'un peptide ou d'un fragment de peptide conforme à l'invention, en mettant en présence ledit échantillon biologique avec ledit/lesdits peptides ou fragment (s) de peptide, auxquels se lient les anticorps anti-Mokola si de tels anticorps sont présents dans l'échantillon biologique à analyser, la lecture du résultat étant révélée par un moyen approprié notamment EIA, RIA, fluorescence. The present invention also relates to an immunological method for detecting anti antibodies peptides and / or peptide fragments of the Mokola virus, which consists in detecting anti-Mokola antibodies possibly present in a biological sample using a peptide or a peptide fragment in accordance with the invention, by bringing together said biological sample with said peptide (s) or peptide fragment (s), to which the anti-Mokola antibodies bind if such antibodies are present in the biological sample to be analyzed, the reading of the result being revealed by an appropriate means, in particular EIA , RIA, fluorescence.
Selon un mode de réalisation avantageux dudit procédé, lesdits peptides ou fragments peptidiques sont fixés sur un support solide approprié. According to an advantageous embodiment of said method, said peptides or peptide fragments are fixed on an appropriate solid support.
Ledit procédé immunologique peut être avantageusement de type direct, de type indirect ou mettre en oeuvre une méthode sandwich ou bi-site. Said immunological method can advantageously be of direct type, of indirect type or implement a sandwich or bi-site method.
Selon un autre mode de réalisation avantageux dudit procédé, lorsque l'on met en oeuvre une méthode de type sandwich, la révélation est réalisée au moyen d'un deuxième anticorps dirigé contre l'anticorps à doser et marqué de manière appropriée. According to another advantageous embodiment of the said method, when a sandwich type method is used, the revelation is carried out by means of a second antibody directed against the antibody to be assayed and appropriately labeled.
Ce procédé permet le titrage en anticorps du sérum et permet notamment de vérifier la séroconversion des individus vaccinés ou de procéder à des enquêtes sérologiques à visée épidémiologique. This process allows the serum antibody titration and in particular makes it possible to verify the seroconversion of vaccinated individuals or to carry out serological surveys for epidemiological purposes.
La présente invention a également pour objet un procédé de détection rapide et spécifique du virus Mokola qui consiste à détecter un virus Mokola, éventuellement présent dans un échantillon biologique à l'aide d'au moins une sonde nucleotidique conforme à l'invention, en mettant en présence ledit échantillon biologique traité de manière appropriée avec ladite/lesdites sondes nucléotidiques, à laquelle/auxquelles se lie l'ARN genomique et/ou les produits de transcription du virus Mokola, si de tels pro
duits sont présents dans l'échantillon, la lecture du résultat étant révélée par un moyen approprié. The subject of the present invention is also a method of rapid and specific detection of the Mokola virus which consists in detecting a Mokola virus, possibly present in a biological sample using at least one nucleotide probe according to the invention, by putting in the presence of said biological sample treated appropriately with said at least one nucleotide probe, to which / to which the genomic RNA and / or the transcripts of the Mokola virus binds, if such pro duits are present in the sample, the reading of the result being revealed by an appropriate means.
La présente invention a également pour objet un kit prêt à l'emploi, pour la mise en oeuvre du procédé conforme à l'invention de détection et/ou d'identification d'au moins un Lyssavirus, caractérisé en ce qu'il comprend outre des quantités utiles de tampons et de réactifs appropriés pour la mise en oeuvre de ladite détection, des doses appropriées d'au moins deux amorces convenables, des doses appropriées d'au moins une sonde nucleotidique et des doses appropriées d'au moins une enzyme de restriction. The subject of the present invention is also a ready-to-use kit for implementing the method according to the invention for detecting and / or identifying at least one Lyssavirus, characterized in that it further comprises useful quantities of buffers and reagents suitable for carrying out said detection, appropriate doses of at least two suitable primers, appropriate doses of at least one nucleotide probe and appropriate doses of at least one enzyme restriction.
La présente invention a également pour objet un kit prêt à l'emploi pour la mise en oeuvre du procédé de détermination dans un échantillon biologique, d'anticorps anti-Mokola, caractérisé en ce qu'il comprend au moins : The subject of the present invention is also a ready-to-use kit for implementing the method for determining in a biological sample, anti-Mokola antibodies, characterized in that it comprises at least:
- des doses appropriées d'au moins un peptide et/ou un fragment peptidique conforme à l'invention ; et - appropriate doses of at least one peptide and / or a peptide fragment in accordance with the invention; and
- des quantités utiles de tampons appropriés pour la mise en oeuvre de ladite détection. - useful quantities of appropriate buffers for the implementation of said detection.
Selon un mode de réalisation avantageux dudit kit, le peptide est de la glycoprotéine G ou un fragment de celle-ci, fixée sur un support solide approprié. According to an advantageous embodiment of said kit, the peptide is glycoprotein G or a fragment thereof, fixed on an appropriate solid support.
Selon un autre mode de réalisation avantageux dudit kit, le peptide est de la nucléoprotéine N ou un fragment de celle-ci, fixée sur un support solide approprié. According to another advantageous embodiment of said kit, the peptide is nucleoprotein N or a fragment thereof, fixed on an appropriate solid support.
Ce kit peut comprendre en outre des doses appropriées d'un deuxième anticorps dirigé contre l'anticorps à doser marqué à l'aide d'une enzyme, d'une substance fluorescente ou d'un isotope radioactif. This kit can also comprise appropriate doses of a second antibody directed against the antibody to be assayed labeled with the aid of an enzyme, a fluorescent substance or a radioactive isotope.
La présente invention a, de plus, pour objet un kit prêt à l'emploi pour la mise en oeuvre du procédé de détection d'un virus Mokola conforme à l'invention, caractérisé en ce qu'il comprend au moins :
- des doses appropriées d'au moins une sonde et/ou fragment de sonde nucleotidique conforme à l'invention ; et The present invention further relates to a ready-to-use kit for implementing the method for detecting a Mokola virus according to the invention, characterized in that it comprises at least: - appropriate doses of at least one probe and / or fragment of nucleotide probe according to the invention; and
- des quantités utiles de tampons appropriés pour la mise en oeuvre de ladite détection. - useful quantities of appropriate buffers for the implementation of said detection.
Outre les dispositions qui précèdent, l'invention comprend encore d'autres dispositions, qui ressortiront de la description qui va suivre, qui se réfère à des exemples de mise en oeuvre de l'invention. In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the invention.
II doit être bien entendu, toutefois, que ces exemples sont donnés uniquement à titre d'illustration de l'objet de l'invention, dont ils ne constituent en aucune manière une limitation. It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.
Exemple 1 : clonage et séquençage de l'ADN complémentaire de l'ARN genomique du virus Mokola. Example 1: Cloning and sequencing of DNA complementary to the genomic RNA of the Mokola virus.
a) Culture du virus a) Culture of the virus
La souche Mokola étudiée a été isolée chez un chat au Zimbabwe. The Mokola strain studied was isolated from a cat in Zimbabwe.
Les virions sont purifiés à partir des plages de lyse obtenues sur une culture de cellules CER. The virions are purified from the lysis plaques obtained on a culture of CER cells.
Le virus Mokola ainsi sélectionné est cultivé sur cellules BHK-21 et purifié selon la méthode décrite par WIKTOR et al. (J. Virol., 1977, 21, 626-635) modifiée. The Mokola virus thus selected is cultured on BHK-21 cells and purified according to the method described by WIKTOR et al. (J. Virol., 1977, 21, 626-635) modified.
b) Purification de l'ARN genomique b) Purification of genomic RNA
Pour isoler l'ARN genomique du virus Mokola, les virions purifiés sont incubés avec 100 μg/ml de protéinase K (Merck) dans du SDS 1,5 %, 100 mM Tris-HCl pH 7,5, 100 mM NaCl, 10 mM EDTA pendant 30 min à 37°C, suivi de deux extractions phénol-chloroforme (vol/vol) et d'une précipitation à l'éthanol.
c) Caractérisation et isolement de clones d'ADNc représentant le gène L et l'extrémité 5' du génome Mokola To isolate the genomic RNA from the Mokola virus, the purified virions are incubated with 100 μg / ml of proteinase K (Merck) in 1.5% SDS, 100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA for 30 min at 37 ° C, followed by two phenol-chloroform extractions (vol / vol) and precipitation with ethanol. c) Characterization and isolation of cDNA clones representing the L gene and the 5 ′ end of the Mokola genome
1.c. Synthèse de l'ADNc 1 C. CDNA synthesis
On synthétise le premier brin d'ADNc en présence de 1 μg de ARN genomique en utilisant un rapport molaire 10 fois supérieur en présence d'une amorce octodécamérique à la fois (l'amorce 3', localisée en position 1-18 ou l'amorce M2, localisée en position 2 901- 2 918 sur le génome rabique, par exemple) en présence de 50 unités de transcriptase réverse, dans un tampon contenant 50 mM de Tris HCl pH 8,3, 8 mM de MgCl2, 100 mM de KCl, 0,8 mM de dATP, 0,8 mM de dGTP, 0,3 mM de dCTP, 0,3 mM de dTTP, 0,2 μM de 32P dCTP, 0,2 μM de 32P dTTP, 30 U/μl de RNasine pendant 2 heures à 42°C. The first strand of cDNA is synthesized in the presence of 1 μg of genomic RNA using a molar ratio 10 times greater in the presence of one octodecameric primer at a time (the 3 'primer, located in position 1-18 or the primer M2, located in position 2,901-2,918 on the rabies genome, for example) in the presence of 50 units of reverse transcriptase, in a buffer containing 50 mM of Tris HCl pH 8.3, 8 mM of MgCl 2 , 100 mM KCl, 0.8 mM dATP, 0.8 mM dGTP, 0.3 mM dCTP, 0.3 mM dTTP, 0.2 μM 32 P dCTP, 0.2 μM 32 P dTTP, 30 U / μl RNasin for 2 hours at 42 ° C.
L'ADNc double brin est préparé selon la méthode de GUBLER et HOFFMAN et séparé selon sa taille sur une colonne Biogel A-50 m. The double stranded cDNA is prepared according to the GUBLER and HOFFMAN method and separated according to its size on a Biogel A-50 m column.
Les fractions (environ 15 à 20 ng d'ADNc) correspondant aux ADNc les plus grands sont insérées au niveau du site Pst I du plasmide pBR322 à l'aide de la méthode d'allongement au dC/dG. The fractions (approximately 15 to 20 ng of cDNA) corresponding to the largest cDNAs are inserted at the Pst I site of the plasmid pBR322 using the dC / dG extension method.
Des souches d'E. Coli DH 5 compétentes sont transformées et les transformants contenant les inserts spécifiques de Mokola sont détectés par hybridation sur filtres de nitrocellulose. Strains of E. Coli DH 5 competent are transformed and the transformants containing the specific Mokola inserts are detected by hybridization on nitrocellulose filters.
2.c. Caractérisation des clones obtenus 2.c. Characterization of the clones obtained
On détermine 3 clones spécifiques de Mokola sélectionnés avec une sonde rage , souche PV (serotype 1) dans la première banque d'ADNc initialisée avec l'amorce M2 : pMB5, pM12 et pMR15a qui couvrent la moitié 5' du génome de Mokola, comme visible sur la figure 1. 3 specific Mokola clones selected with a rabies probe, strain PV (serotype 1) are determined in the first cDNA library initialized with the primer M2: pMB5, pM12 and pMR15a which cover the 5 'half of the Mokola genome, as visible in Figure 1.
La banque d'ADNc obtenue avec l'amorce 3' permet de sélectionner le clone pMA10 avec des sondes Mokola issues du premier clonage. A partir de celui-ci, le clone pMD10 est sélectionné.
Les inserts pMR15a et pMD10 ont été séquences et atteignent respectivement les extrémités 5' et 3' de l'ARN genomique, prouvant que les cinq clones ci-dessus mentionnés suffisent à couvrir la totalité du génome du virus Mokola. The cDNA library obtained with the 3 'primer makes it possible to select the clone pMA10 with Mokola probes originating from the first cloning. From this, the pMD10 clone is selected. The inserts pMR15a and pMD10 have been sequenced and reach the 5 'and 3' ends of the genomic RNA respectively, proving that the five clones mentioned above are sufficient to cover the entire genome of the Mokola virus.
La figure 2 compare les extrémités 3' et 5' du génome du virus Mokola avec celles de la souche PV du génome rabique. Figure 2 compares the 3 'and 5' ends of the genome of the Mokola virus with those of the PV strain of the rabies genome.
Les nucléotides complémentaires entre les deux extrémités sont spécifiées à l'aide de longues lignes verticales. Les séquences consensus spécifiant le début de l'ARNm du gène N, l'arrêt de l'ARNm du gène L et le signal de début de la protéine N sont indiquées. The complementary nucleotides between the two ends are specified using long vertical lines. The consensus sequences specifying the start of the N gene mRNA, the stop of the L gene mRNA and the start signal of the N protein are indicated.
Les nucléotides des extrémités génomiques 3' et 5' du virus Mokola qui sont identiques à ceux de la souche PV sont spécifiés à l'aide de lignes verticales courtes. Les résidus sont numérotés à partir des extrémités génomiques. The nucleotides of the 3 'and 5' genomic ends of the Mokola virus which are identical to those of the PV strain are specified using short vertical lines. The residues are numbered from the genomic ends.
La figure 3 montre l'hybridation hétérologue et homologue de sondes rabiques localisées sur les gènes FIG. 3 shows the heterologous and homologous hybridization of rabies probes located on the genes
N et L avec des Northern blots de cellules infectées par le virus Mokola ou le virus rabique respectivement. L'ARN cytoplasmique total, obtenu à partir de cellules BHK-21 est prélevé, 6, 12, 24 ou 48 heures après l'infection avec le virus Mokola (traces 1) ou l'infection avec le virus rabique (trace 2) ; la trace 3 correspondant à un témoin négatif (cellules non infectées), puis est soumis à une électrophorèse sur gel d'agarose 1,2 %- formaldéhyde. Les ARN séparés sont déposés sur des membranes en nylon et hybrides avec une sonde marquée auN and L with Northern blots of cells infected with the Mokola virus or the rabies virus respectively. Total cytoplasmic RNA, obtained from BHK-21 cells, is collected 6, 12, 24 or 48 hours after infection with the Mokola virus (traces 1) or infection with the rabies virus (trace 2); trace 3 corresponding to a negative control (uninfected cells), then is subjected to electrophoresis on 1.2% agarose gel - formaldehyde. The separated RNAs are deposited on nylon and hybrid membranes with a probe labeled with
32P, constituée soit par la séquence 377-1039 de l'ADNc du génome rabique (sonde N : A), soit par la séquence 32 P, consisting either of the sequence 377-1039 of the cDNA of the rabies genome (probe N: A), or of the sequence
7034-7134 (sonde L : B) de l'ADNc du génome rabique.
Exemple 2 : Caractérisation des ARNm de Mokola synthétisés in vivo. 7034-7134 (probe L: B) of the cDNA of the rabies genome. Example 2: Characterization of the Mokola mRNAs synthesized in vivo.
Les sondes d'ADNc sont sélectionnées conformément à la figure 1 pour caractériser les produits de transcription in vivo lors d'une infection par le virus Mokola de cellules BHK-21. Six différents transcrits sont observés par l'analyse en Northern blot, comme visible sur la figure 4 qui montre les ARNm du virus Mokola s'hybridant avec les ADNc N, N + M1, M1 + M2, G et L du génome du virus Mokola. The cDNA probes are selected in accordance with FIG. 1 to characterize the transcripts in vivo during an infection with the Mokola virus of BHK-21 cells. Six different transcripts are observed by the Northern blot analysis, as visible in FIG. 4 which shows the mRNAs of the Mokola virus hybridizing with the cDNAs N, N + M1, M1 + M2, G and L of the genome of the Mokola virus .
L'ARN cytoplasmique total de virus Mokola, obtenu à partir de cellules BHK-21 infectées, est dénaturé en présence de formamide 50 % et des échantillons de 15 μg sont soumis à une électrophorèse sur gel d'agarose 1 % contenant du formaldéhyde. L'ARN séparé est déposé sur des membranes de nylon avec des sondes d'ADNc marquées au 32P (N, M1 + M2, N + M1, G et L). Les flèches indiquent les positions de l'ARN ribosomal 18 S et 28 S, révélés par une coloration au bromure d'éthidium et l'identité des différents ARNm est révélée par autoradiographie. The total cytoplasmic RNA of Mokola virus, obtained from infected BHK-21 cells, is denatured in the presence of 50% formamide and 15 μg samples are subjected to electrophoresis on 1% agarose gel containing formaldehyde. The separated RNA is deposited on nylon membranes with cDNA probes labeled with 32 P (N, M1 + M2, N + M1, G and L). The arrows indicate the positions of the 18 S and 28 S ribosomal RNA, revealed by staining with ethidium bromide and the identity of the different mRNAs is revealed by autoradiography.
La caractérisation et la taille de ces transcrits Mokola, montrent que la carte transcriptionnelle du virus Mokola est identique à celle du virus rabique. En fait 5 ARNm monocistroniques apparaissent successivement à partir de l'extrémité 3' du génome. Leur longueur est estimée, sur gel d'agarose, à 1750, 1250, 1000, 2400 nucléotides, y compris la queue polyadénylée. Ils codent pour les protéines N, M1, M2 et G respectivement. Seul le cinquième, dont la longueur est estimée à 4800 nucléotides et codant pour la protéine L, est inférieur à la valeur prévue conformément à la longueur du génome. Un sixième produit transcriptionnel est visible et correspond à un ARNm M1-M2 bicistronique (ligne M1 + M2). Sa longueur est estimée à 2200 nucléotides.
Exemple 3 Procédé de détermination de la glycoprotéine G de virus Mokola dans une préparation ou un liquide biologique en présence d'un anticorps antiglycoprotéine 6 conforme à l'invention conjugué à la peroxydase de raifort. The characterization and the size of these Mokola transcripts show that the transcriptional map of the Mokola virus is identical to that of the rabies virus. In fact, 5 monocistronic mRNAs appear successively from the 3 'end of the genome. Their length is estimated, on agarose gel, at 1750, 1250, 1000, 2400 nucleotides, including the polyadenylated tail. They code for proteins N, M1, M2 and G respectively. Only the fifth, whose length is estimated at 4800 nucleotides and coding for the L protein, is less than the value predicted in accordance with the length of the genome. A sixth transcription product is visible and corresponds to a bicistronic M1-M2 mRNA (line M1 + M2). Its length is estimated at 2200 nucleotides. Example 3 Method for determining the glycoprotein G of Mokola virus in a preparation or a biological liquid in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated with horseradish peroxidase.
La suspension est clarifiée par centrifugation à 1500 x g pendant 30 min à 4°C. Les surnageants clarifiés de chaque échantillon sont distribués en double dans les puits de plaques de microtitration sensibilisées avec des anticorps anti-glycoprotéine G conformes à l'invention (200 μl/puits). Les microplaques sont alors incubées pendant une heure à 37°C. Après des lavages répétés avec un tampon PBS-Tween, chaque puits reçoit 200 μl d'anticorps anti-glycoprôtéine G conjugué à la peroxydase de raifort. Les microplaques sont alors incubées pendant une heure à 37°C, puis à nouveau lavées. L'antigène viral est alors quantifié par l'apparition d'une coloration, lorsque le substrat de la peroxydase est ajouté ; le substrat est un mélange d'o-phénylènediamine et peroxyde d'hydrogène (200 μl/puits). Les microplaques sont laissées 20 min à la température ambiante pour permettre à la coloration de se développer. On arrête la réaction en ajoutant du H2SO4N (50 μl/puits). On mesure la coloration au spectrophotomètre. The suspension is clarified by centrifugation at 1500 xg for 30 min at 4 ° C. The clarified supernatants from each sample are distributed in duplicate in the wells of microtitration plates sensitized with anti-glycoprotein G antibodies in accordance with the invention (200 μl / well). The microplates are then incubated for one hour at 37 ° C. After repeated washing with PBS-Tween buffer, each well receives 200 μl of anti-glycoprotein G antibody conjugated with horseradish peroxidase. The microplates are then incubated for one hour at 37 ° C, then washed again. The viral antigen is then quantified by the appearance of a coloration, when the substrate of the peroxidase is added; the substrate is a mixture of o-phenylenediamine and hydrogen peroxide (200 μl / well). The microplates are left 20 min at room temperature to allow the staining to develop. The reaction is stopped by adding H 2 SO 4 N (50 μl / well). The coloration is measured with a spectrophotometer.
Exemple 4 : Procédé de détermination de la glycoprotéine G de virus Mokola en présence d'un anticorps anti-glycoprotéine 6 conforme à l'invention conjugué à la l'isothiocyanate de fluorescéine. Example 4: Method for determining the glycoprotein G of Mokola virus in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated to fluorescein isothiocyanate.
Des cultures cellulaires infectées par le virus Mokola sont fixées pendant 30 min dans l'acétone froide. La coloration est réalisée en couvrant les lames pendant 30 min à 37°C avec des anticorps anti-glycoprotéine G de virus Mokola conjugués à l'isothiocyanate de fluorescéine. Le bleu Evans (1/5000) est utilisé comme contre-colorant. Les lames sont lavées par immersion dans du PBS à pH 7,6 pendant 5 min. Un milieu de montage à la
glycérine est utilisé et les lames sont examinées au microscope à épifluorescence. Cell cultures infected with the Mokola virus are fixed for 30 min in cold acetone. Staining is carried out by covering the slides for 30 min at 37 ° C. with anti-glycoprotein G antibodies of Mokola virus conjugated to fluorescein isothiocyanate. Evans blue (1/5000) is used as a counter-dye. The slides are washed by immersion in PBS at pH 7.6 for 5 min. A mounting medium at the glycerin is used and the slides are examined under an epifluorescence microscope.
Exemple 5 : Procédé de détection et de titrage des anticorps anti-glycoprotéine 6 du virus Mokola, dans le sang des individus vaccinés, avec des peptides et/ou fragments de peptides conformes à l'invention. Example 5: Method for detecting and titrating anti-glycoprotein 6 antibodies of the Mokola virus, in the blood of vaccinated individuals, with peptides and / or fragments of peptides in accordance with the invention.
Ce test repose qur l'utilisation d'une phase solide, sur laquelle est fixée la glycoprotéine du virus, et d'un conjugué enzymatique, protéine A de Staphylococeus aureus couplée à la peroxydase. This test is based on the use of a solid phase, on which the virus glycoprotein is attached, and of an enzyme conjugate, protein A of Staphylococeus aureus coupled with peroxidase.
On effectue des dilutions, de raison deux, des sérums de contrôle positifs et négatifs. Les sérums ou plasmas inconnus sont dilués au l/100è. 100 μl de ces différents sérums sont déposées dans chaque cupule. Two-fold dilutions are made of positive and negative control sera. Unknown sera or plasmas are diluted to 1/100. 100 μl of these different sera are deposited in each well.
On couvre d'un film autocollant et on incube les plaques dans une étuve pour microplaque thermostatée, pendant 60 min à 37°C. Cover with self-adhesive film and incubate the plates in a thermostatically controlled microplate oven for 60 min at 37 ° C.
On retire le film adhésif ; on aspire le contenu de chaque plaque, on effectue trois lavages sucessifs des cupules puis on sèche les plaques. The adhesive film is removed; the contents of each plate are aspirated, three successive washings of the wells are carried out, then the plates are dried.
On distribue 100 μl de la solution de conjugué dans toutes les cupules et on incube les plaques, recouvertes d'un film adhésif, pendant 60 min à 37°C. 100 μl of the conjugate solution are distributed in all the wells and the plates are incubated, covered with an adhesive film, for 60 min at 37 ° C.
On aspire le contenu des cupules, on les lave quatre fois puis on sèche les plaques. The contents of the wells are aspirated, washed four times and then the plates are dried.
On distribue 100 μl de la solution de révélation (tampon et substrat de la peroxydase) dans chaque cupule et on laisse la réaction se développer à l'obscurité pendant 30 min à température ambiante. 100 μl of the development solution (buffer and peroxidase substrate) are distributed in each well and the reaction is allowed to develop in the dark for 30 min at room temperature.
On ajoute 50 μl de la solution d'arrêt puis on lit la densité optique des cupules à 492 nm. 50 μl of the stop solution are added and then the optical density of the wells is read at 492 nm.
La comparaison des densités optiques des sérums inconnus, avec la courbe d'étallonage du contrôle positif titré en Unités Internationales par ml, permet d'affecter à chacun un titre en Unités Equivalentes par ml.
Exemple 6 : Sonde nucleotidique conforme à l'invention. The comparison of the optical densities of the unknown sera, with the standardization curve of the positive control titrated in International Units per ml, makes it possible to assign to each a title in Equivalent Units per ml. Example 6: Nucleotide probe according to the invention.
Il s'agit de sondes en simple brin de sens genomique (sens -) ou antigénomique (sens +) clones dans le vecteur M13. These are single-strand probes with genomic (sense -) or antigenomic (sense +) direction cloned into the vector M13.
3,5 μl (environ 0,5 picomoles) de matrice M13 clonée sont mis en présence de 1 μl (0,5 picomoles) d'amorce universelle et de 0,5 μl d'un tampon comprenant lOmM Tris-HCl pH 7,5, 10mM MgCl2, 50mM NaCl, ImM DTT. Le mélange, fermé hermétiquement, est plongé dans un bainmarie qui est amené à ébullition puis laissé à refroidir progressivement sur la paillasse. Au bout d'1/2 h, l'eau est à 40°C et l'hybridation est faite. 3.5 μl (approximately 0.5 picomoles) of cloned M13 matrix are placed in the presence of 1 μl (0.5 picomoles) of universal primer and 0.5 μl of a buffer comprising 10 mM Tris-HCl pH 7, 5, 10mM MgCl 2 , 50mM NaCl, ImM DTT. The mixture, hermetically closed, is immersed in a bain-marie which is brought to a boil and then allowed to cool gradually on the bench. After 1/2 h, the water is at 40 ° C and the hybridization is done.
Les 5 μl de matrice hybridée sont alors complétée par : The 5 μl of hybridized matrix is then completed by:
- 2 X (4 μl d'un 32P-α-dNTP) 4000Ci/mmole, lmCi/ml ; - 2 X (4 μl of a 32 P-α-dNTP) 4000Ci / mmole, lmCi / ml;
- 1 μl d'un mélange des 4 dNTP à 125 picomoles/μl chacun ; - 1 μl of a mixture of the 4 dNTPs at 125 picomoles / μl each;
- 1 μl (environ 1 U) d'E. Coli polymérase - 1 μl (approximately 1 U) of E. Coli polymerase
(fragment de Kleenow). (fragment of Kleenow).
La réaction se déroule pendant 20 min à +37°C, puis est stoppée par un chauffage de 3 min à + 65°C. The reaction takes place for 20 min at + 37 ° C, then is stopped by heating for 3 min at + 65 ° C.
A ce moment de la manipulation, la matrice M13, copiée par la polymérase à partir de l'amorce universelle, est en double brin sur une longueur dépendant de la quantité de mononucléotides introduite initialement dans le milieu. De plus, cette longueur varie statistiquement d'un clone à l'autre. Pour homogénéiser la taille de la sonde, on choisit de couper au niveau d'un site de restriction unique situé dans une position suffisamment proche de l'amorce pour que le vecteur soit en double brin à son niveau. At this time of manipulation, the M13 matrix, copied by the polymerase from the universal primer, is in double strand over a length depending on the amount of mononucleotides initially introduced into the medium. In addition, this length varies statistically from one clone to another. To homogenize the size of the probe, we choose to cut at a single restriction site located in a position close enough to the primer so that the vector is in double strand at its level.
Pour ce faire, le mélange réactionnel de synthèse (15 μl) est amené à une concentration saline adaptée à l'enzyme de restriction choisie (MANIATIS et al.,
1982), et la coupure se déroule pendant 1 h, à la température appropriée, dans un volume final de 20 μl. Un volume identique d'un tampon de dépôt dénaturant est alors ajouté. Après dénaturation thermique, le mélange est déposé sur un gel d'acrylamide-urée dénaturant à 6 % (0,5 mm d'épaisseur). La migration dure 1 heure à 1200 volts. La sonde radioactive est repérée par autoradiographie puis découpée. Pour les sondes dont la taille n'excède pas 400 bases, une agitation durant 1 h 30 min dans un tampon adapté (NH4COOH 0,5 M, MgCl2 10mM, EDTA ImM) est suffisante pour éluer 80 % de la radioactivité. Au-delà de 400 bases, il est plus prudent de pratiquer une électroélution (MANIATIS et al., 1982). L'éluat est consécutivement extrait par un volume égal de phénol saturé avant que la sonde ne soit précipitée classiquement à l'éthanol. Après deux lavages dans l'éthanol à 70 % et un séchage, le précipité est repris dans 20 μl d'eau. On obtient de manière routinière des sondes marquées de 4 000 à 10 000 cpm (cerenkof)/μl. To do this, the synthetic reaction mixture (15 μl) is brought to a salt concentration suitable for the restriction enzyme chosen (MANIATIS et al., 1982), and the cut takes place for 1 h, at the appropriate temperature, in a final volume of 20 μl. An identical volume of a denaturing deposit buffer is then added. After thermal denaturation, the mixture is deposited on a 6% denaturing acrylamide-urea gel (0.5 mm thick). The migration lasts 1 hour at 1200 volts. The radioactive probe is identified by autoradiography and then cut out. For probes whose size does not exceed 400 bases, stirring for 1 h 30 min in a suitable buffer (NH 4 COOH 0.5 M, MgCl 2 10 mM, EDTA ImM) is sufficient to elute 80% of the radioactivity. Beyond 400 bases, it is more prudent to practice electroelution (MANIATIS et al., 1982). The eluate is consecutively extracted with an equal volume of saturated phenol before the probe is conventionally precipitated with ethanol. After two washes in 70% ethanol and drying, the precipitate is taken up in 20 μl of water. Probes marked with 4,000 to 10,000 cpm (cerenkof) / μl are routinely obtained.
Cette sonde peut alors être utilisée pour l'hybridation directe sur les blots ou pour la cartographie à la nucléase S1. Les techniques que nous suivons sont classiques bien que légèrement modifiées. Voici quelques précisions : This probe can then be used for direct hybridization on blots or for S1 nuclease mapping. The techniques we follow are classic, although slightly modified. Here are some details:
Exemple 7 : détection d'un ARN de virus Mokola à l'aide d'une sonde conforme à l'invention (Blots d'ARN) Example 7: Detection of a Mokola virus RNA using a probe in accordance with the invention (RNA blots)
Un milieu d'hybridation qui comprend : A hybridization medium which includes:
- un mélange Na1H2PO4-Na2H1PO4 pH 7,4 0,5 M- a mixture of Na 1 H 2 PO 4 -Na 2 H 1 PO 4 pH 7.4 0.5 M
- SDS 7 % - EDTA 1 mM- SDS 7% - EDTA 1 mM
- sérum albumine bovine (BSA) 1 %, convient à la fois pour la préhybridation (au maximum 2 h) et pour l'hybridation (en général durant la nuit) qui se déroulent toutes deux à + 65°C. Le lavage des filtres est réalisé à la même température, en quatre bains suc
cessifs de 10 min chacun, dans le tampon suivant : - bovine serum albumin (BSA) 1%, suitable for both prehybridization (maximum 2 h) and for hybridization (generally overnight) which both take place at + 65 ° C. The filters are washed at the same temperature, in four suction baths stops of 10 min each, in the following buffer:
- un mélange Na1H2PO4-Na2H1PO4 pH 7,4 40 mM- a mixture of Na 1 H 2 PO 4 -Na 2 H 1 PO 4 pH 7.4 40 mM
- SDS 1 %- SDS 1%
- EDTA pH 7,5 1 mM Pour des hybridations de sondes strictement complémentaires aux transcrits recherchés, cette méthode donne de forts bons résultats, en particulier une très grande discrétion du bruit de fond. - EDTA pH 7.5 1 mM For hybridizations of probes strictly complementary to the transcripts sought, this method gives very good results, in particular very great discretion of the background noise.
Exemple 8 : Procédé d'identification rapide (typage) de faibles quantités de Lyssavirus . Example 8: Method for rapid identification (typing) of small quantities of Lyssavirus.
Environ 1 μg d'ARN total provenant de cellules fibroblastiques BHK21 ou neuronales murines N2A infectées par un Lyssavirus (souches rabiques classiques Pasteur, PV, CVS, Av01, PM, ERA, souches rabiques sauvages "chien du Gabon", "chauve-souris brésilienne", "renard d'Europe", Mokola, etc...), est hybridée avec environ 100 ng d'amorce "G", correspondant à un oligonucléotide long de 23 nucléotides, de sens (+) et s 'étendant entre les positions 4665 et 4687 du génome rage PV, et un ADN de sens (+) complémentaire au génome est amorcé spécifiquement et synthétisé comme décrit dans l'exemple 1. Le milieu réactionnel est extrait au phénol, précipité à l'éthanol, lavé deux fois avec de l'éthanol à 70 %, puis séché. Il est alors repris dans 50 μl comprenant : Approximately 1 μg of total RNA from BHK21 fibroblastic cells or N2A murine neuronal cells infected with a Lyssavirus (classic Rabies strains Pasteur, PV, CVS, Av01, PM, ERA, wild rabies strains "Gabon dog", "Brazilian bat "," European fox ", Mokola, etc ...), is hybridized with approximately 100 ng of primer" G ", corresponding to an oligonucleotide long 23 nucleotides, of direction (+) and extending between positions 4665 and 4687 of the rage PV genome, and a DNA of sense (+) complementary to the genome is specifically initiated and synthesized as described in Example 1. The reaction medium is extracted with phenol, precipitated with ethanol, washed twice with 70% ethanol, then dried. It is then taken up in 50 μl comprising:
- Amorce "L" 100 ng - Primer "L" 100 ng
- Tris HCl, pH 8,8 60 mM - Tris HCl, pH 8.8 60 mM
- Ammonium sulfate 17 mM - Ammonium sulfate 17 mM
- MgC12 6,7 mM - MgC12 6.7 mM
- B-mercaptoéthanol 10 mM - B-mercaptoethanol 10 mM
- EDTA 5 μM - EDTA 5 μM
- BSA 170 μg/ml - BSA 170 μg / ml
- DMSO 10 % (vol/vol)- DMSO 10% (vol / vol)
- dNTP's 25 mM - dNTP's 25 mM
- Taq DNA polymérase 2 Unités ;
l'amorce "L" correspond à un oligonucléotide long de 24 nucléotides, de sens (-) et s 'étendant entre les positions 5520 et 5543 du génome rage PV. - Taq DNA polymerase 2 Units; the primer "L" corresponds to an oligonucleotide 24 nucleotides long, of direction (-) and extending between positions 5520 and 5543 of the PV rabies genome.
L'amorce G s'hybride avec la région 4675-4697 de la séquence nucleotidique (I) ci-dessus, laquelle amorce G présente une homologie de l'ordre de 80 % avec la région 4675-4697 de la séquence (I) du virus Mokola. The primer G hybridizes with the region 4675-4697 of the nucleotide sequence (I) above, which primer G has a homology of the order of 80% with the region 4675-4697 of the sequence (I) of the Mokola virus.
L'amorce L s 'hybride spécifiquement avec la région 5545-5568 de la séquence nucleotidique de formule (I) du virus Mokola. Primer L hybridizes specifically with region 5545-5568 of the nucleotide sequence of formula (I) of the Mokola virus.
Le milieu réactionnel est placé dans un tube eppendorf de 1,5 ml, recouvert de 100 μl d'huile minérale pour éviter 1 'evaporation, et incubé dans une "machine à P.C.R." lui faisant subir les étapes suivantes : The reaction medium is placed in a 1.5 ml eppendorf tube, covered with 100 μl of mineral oil to avoid evaporation, and incubated in a "P.C.R. machine" subjecting him to the following stages:
+ 95°C 5 min pour dénaturer + 95 ° C 5 min to denature
+ 50°C 2 min pour que les amorces + 50 ° C 2 min so that the primers
s'hybrident hybridize
+ 72°C 2 min pour que la Taq polymérase + 72 ° C 2 min for the Taq polymerase
allonge les brins d'ADNc. lengthens the strands of cDNA.
Cette étape est réalisée une fois, puis elle est répétée 40 fois de suite en limitant le temps de dénaturation à 2 min. This step is carried out once, then it is repeated 40 times in succession, limiting the denaturation time to 2 min.
Enfin, elle est répétée une 41ème fois allongeant le temps de synthèse des ADNc à 10 min pour compléter la réaction. Finally, it is repeated a 41st time extending the synthesis time of the cDNAs to 10 min to complete the reaction.
La bande d'ADNc amplifiée peut être observée après migration des produits réactionnels sur un gel d'agarose approprié. Après transfert, elle peut être caractérisée au moyen d'une sonde appropriée, provenant du clonage du génome de la souche analysée ou d'une autre souche de Lyssavirus . The amplified cDNA band can be observed after migration of the reaction products on an appropriate agarose gel. After transfer, it can be characterized by means of an appropriate probe, originating from the cloning of the genome of the strain analyzed or of another strain of Lyssavirus.
Ces amorces ont été testées avec succès sur toutes les souches rabiques fixes ou sauvages disponibles ainsi que sur le virus Mokola comme le montre la figure 7 ; elles permettent notamment d'amplifier le pseudogène psi de n'importe quel Lyssavirus. La région amplifiée.
d'environ 860 nucléotides, peut être ensuite caractérisée : These primers have been successfully tested on all available fixed or wild rabies strains as well as on the Mokola virus as shown in FIG. 7; they make it possible in particular to amplify the psi pseudogen of any Lyssavirus. The amplified region. approximately 860 nucleotides, can then be characterized:
- soit par hybridation avec une sonde radioactive plus ou moins spécifique de souche de Lyssavirus, - either by hybridization with a radioactive probe more or less specific for the Lyssavirus strain,
- soit par hydrolyse avec des enzymes de restriction, plus ou moins spécifiques de souches, - either by hydrolysis with restriction enzymes, more or less specific for strains,
- soit encore par la séquence directe et la carte de restriction déduite de la région concernée, pour typer et identifier les souches de Lyssavirus présentes dans un échantillon dans les 12 heures sans avoir recours à l'immunologie. - either by the direct sequence and the restriction map deduced from the region concerned, to type and identify the strains of Lyssavirus present in a sample within 12 hours without resorting to immunology.
La figure 7 correspond à un gel d'électrophorèse et comporte les 16 traces suivantes : Figure 7 corresponds to an electrophoresis gel and has the following 16 traces:
1 : Marqueur de taille 1: Size marker
2 à 9 : Différentes souches rabiques : virus de la rage souche PM, virus de la rage souche AVO1, virus de la rage souche CVS, virus de la rage souche ERA, virus de la rage souche PV, virus de la rage souche Pasteur, virus rage Sauvage (ovin), virus rage Sauvage (ovin) 2 to 9: Different rabies strains: rabies strain PM strain, rabies strain AVO1, rabies strain CVS, rabies strain ERA, rabies strain PV, rabies strain Pasteur, Wild rabies virus (sheep), Wild rabies virus (sheep)
10 : Virus Mokola 10: Mokola virus
11 à 15 : Divers témoins positifs et négatifs 16 : Marqueur de taille 11 to 15: Various positive and negative controls 16: Size marker
La bande d'ADNc amplifiée peut en outre être hydrolysée par des enzymes de restriction sélectionnées capables de différencier rapidement : The amplified cDNA band can also be hydrolyzed by selected restriction enzymes capable of quickly differentiating:
- les différentes souches rabiques fixes ; - the different fixed rabies strains;
- les souches rabiques sauvages qui sont présentent actuellement en France ; - wild rabies strains which are present in France;
- le virus Mokola. - the Mokola virus.
Les huit enzymes suivantes : BamH I, BstX I, The following eight enzymes: BamH I, BstX I,
Fnu4H I, Hind II, Hind III, Pst I, Rsa I, Taq I permettent de typer les différentes souches comme suit : Fnu4H I, Hind II, Hind III, Pst I, Rsa I, Taq I allow the different strains to be typed as follows:
Dans un premier temps, on utilise une série de 4 enzymes pour distinguer la provenance du fragment amplifié variable délimité par les amorces G et L précitées, comme visible sur le Tableau I ci-après.
Firstly, a series of 4 enzymes is used to distinguish the source of the variable amplified fragment delimited by the aforementioned primers G and L, as visible in Table I below.
Dans un deuxième temps, on peut affiner la distinction à l'aide des enzymes suivantes : In a second step, we can refine the distinction using the following enzymes:
Il ressort de ce Tableau II que Rsa I sépare qualitativement PV de ERA-SAD et que Taq I sépare quantativement ERA de SAD. It appears from this Table II that Rsa I qualitatively separates PV from ERA-SAD and that Taq I quantitatively separates ERA from SAD.
Les deux autres enzymes sont plus spécifiques de groupes : The other two enzymes are more group specific:
. BstX I ne coupe que PV, ERA et SAD ;
. Fnu4H I ne coupe que les souches rabiques sauvages et le virus Mokola. . BstX I only cuts PV, ERA and SAD; . Fnu4H I only cuts wild rabies strains and the Mokola virus.
La figure 8 montre que les bandes amplifiées provenant des souches ERA et CVS sont respectivement et spécifiquement coupées par Bstx I et BamH I. FIG. 8 shows that the amplified bands originating from the ERA and CVS strains are respectively and specifically cut by Bstx I and BamH I.
Exemple 9 : Procédé de détection rapide (réponse Oui/Non) de faibles quantités de Lyssavirus. Example 9: Method for rapid detection (Yes / No response) of small amounts of Lyssavirus.
Les amorces Na et Nb telles que définies dans l'invention, permettent, par exemple, l'amplification de la zone 587-1029 de la séquence nucleotidique de formule (I) du virus Mokola qui est une zone conservée d'un Lyssavirus ; ce procédé de détection rapide de faibles quantités de Lyssavirus dans un échantillon conforme à l'invention, a l'avantage de permettre de porter un diagnostic d'infection à Lyssavirus sur des infections débutantes ou sur des prélèvements de salive effectués chez les animaux domestiques suspects de contamination humaine, plusieurs jours avant leur mort, contrairement à la pratique actuelle ; en effet, les amorces Na et Nb telles que définies dans l'invention, situées sur le gène N et peu distantes l'une de l'autre (400 pb) permettent la détection (réponse Oui/Non) d'un Lyssavirus même présent en très petites quantités et ce, même si l'échantillon est très dégradé. En effet, la sensibilité des techniques habituelles est relativement faible par rapport à la méthode conforme à l'invention, qui est rapide et permet de porter un diagnostic en moins de 12 heures. The primers Na and Nb as defined in the invention allow, for example, the amplification of the 587-1029 region of the nucleotide sequence of formula (I) of the Mokola virus which is a conserved region of a Lyssavirus; this method of rapid detection of small amounts of Lyssavirus in a sample in accordance with the invention has the advantage of making it possible to make a diagnosis of infection with Lyssavirus on early infections or on saliva samples taken from suspect domestic animals human contamination, several days before their death, contrary to current practice; indeed, the primers Na and Nb as defined in the invention, located on the N gene and not far apart from one another (400 bp) allow the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
Exemple 10 : Expression de la glycoprotéine du virus Mokola. Example 10 Expression of the Mokola virus glycoprotein.
Les inserts pMD10 et pMA10, issus du clonage du génome Mokola, couvrent chacun pour moitié le gène de la glycoprotéine G. Grâce au site de restriction BglI qu'ils ont en commun, on peut les rejoindre en un seul insert pM7 couvrant les 6 830 nucléotides 3' du génome. partir de celui-ci, on isole l'ADNc du gène de la glyco
protéine G par une double digestion combinant les enzymes de restriction FnuDII et Sspl, qui est inséré entre la région promotrice 5' du gène de la polyédrine du baculovirus et sa région 3' correspondant notamment au signal de polyadénylation. Cette construction est réalisée dans un vecteur pUC, pour permettre sa multiplication. Elle est ensuite cotransfectée avec un baculovirus de type sauvage dans des cellules de chenille Spodoptera frugiperda Sf9 se multipliant en suspension. Le gène de la glycoprotéine G s'insère en lieu et place de celui de la polyédrine du virus sauvage, par double recombinaison. Après clonage, les plages de baculovirus recombinants sont repérées par leur aspect terne en épiluminescence, qui se distingue aisément de l'aspect réfringent des plages de virus sauvage. The inserts pMD10 and pMA10, derived from the cloning of the Mokola genome, each cover half of the glycoprotein G gene. Thanks to the restriction site BglI which they have in common, they can be joined in a single insert pM7 covering the 6,830 nucleotides 3 'of the genome. from this, we isolate the cDNA from the glyco gene protein G by a double digestion combining the restriction enzymes FnuDII and Sspl, which is inserted between the promoter region 5 ′ of the baculovirus polyhedrin gene and its region 3 ′ corresponding in particular to the polyadenylation signal. This construction is carried out in a vector pUC, to allow its multiplication. It is then cotransfected with a wild-type baculovirus in spodoptera frugiperda Sf9 caterpillar cells multiplying in suspension. The G glycoprotein gene is inserted in place of that of the wild virus polyhedrin, by double recombination. After cloning, the recombinant baculovirus plaques are identified by their dull appearance in epiluminescence, which is easily distinguished from the refractive aspect of the wild virus plaques.
Quelques clones recombinants sélectionnés ont servi à réinfecter des cultures de cellules de chenille. Au bout de trois jours après l'infection, les extraits cellulaires protéiques totaux ont été préparé. Une bande protéique supplémentaire, de taille voisine de celle de la glycoprotéine Mokola a été observée uniquement dans les extraits provenant des clones recombinants. Afin de mieux étudier cette protéine supplémentaire, d'autres cultures ont été infectées dans un milieu carence en méthionine, puis complémenté avec de la méthionine radioactive. La glycoprotéine G est alors apparue de façon beaucoup plus intense, représentant sans aucun doute le polypeptide le plus fortement exprimé dans les cultures au moment du marquage (figure 5a). A few selected recombinant clones were used to reinfect caterpillar cell cultures. After three days after infection, the total protein cell extracts were prepared. An additional protein band, of size close to that of the Mokola glycoprotein was observed only in the extracts coming from the recombinant clones. In order to better study this additional protein, other cultures were infected in a methionine deficiency medium, then supplemented with radioactive methionine. The glycoprotein G then appeared in a much more intense manner, undoubtedly representing the polypeptide most strongly expressed in the cultures at the time of labeling (FIG. 5a).
La figure 5b correspond à un témoin négatif. Figure 5b corresponds to a negative control.
La mise en évidence de l'expression de la glycoprotéine du virus Mokola à la surface des cellules infectées par le Baculovirus recombinant est effectuée sur des cellules de Spodoptera frugiperda, après infection par le baculovirus recombinant exprimant la glycoprotéine du virus Mokola. Les cellules sont fixées
dans l'acétone à - 20ºC pendant 30 min. Elles sont ensuite incubées avec les Acm dirigés contre la glycoprotéine du virus Mokola. Demonstration of the expression of the Mokola virus glycoprotein on the surface of cells infected with the recombinant Baculovirus is carried out on Spodoptera frugiperda cells, after infection with the recombinant baculovirus expressing the Mokola virus glycoprotein. Cells are fixed in acetone at - 20ºC for 30 min. They are then incubated with mAbs directed against the Mokola virus glycoprotein.
Après lavage, la révélation se fait par incubation avec un anticorps anti-immunoglobuline de souris couplé à l'isothiocyanate de fluorescéine (ITCF). After washing, the revelation is carried out by incubation with a mouse anti-immunoglobulin antibody coupled to fluorescein isothiocyanate (ITCF).
L'observation après lavage se fait au microscope sous excitation ultra-violette (figures 5a et 5b). The observation after washing is done under the microscope under ultraviolet excitation (Figures 5a and 5b).
La glycoprotéine a été également clairement localisée à la surface des cellules infectées par le virus recombinant, au moyen d'anticorps monoclonaux fluorescents spécifiques de la glycoprotéine du virus Mokola (figure 6). Sur cette figure 6, les traces 1, 2 et 3 correspondent à des cellules de Spodoptera frugiperda après infection par le baculovirus recombinant exprimant la glycoprotéine G du virus Mokola : la flèche montre l'emplacement de la glycoprotéine G sur le gel ; La trace T correspond à un témoin négatif. The glycoprotein was also clearly localized on the surface of the cells infected with the recombinant virus, by means of fluorescent monoclonal antibodies specific for the glycoprotein of the Mokola virus (FIG. 6). In this FIG. 6, traces 1, 2 and 3 correspond to Spodoptera frugiperda cells after infection with the recombinant baculovirus expressing the glycoprotein G of the Mokola virus: the arrow shows the location of the glycoprotein G on the gel; Trace T corresponds to a negative control.
Exemple 11 : Mise en évidence du pouvoir immunogène de la glycoprotéine du virus Mokola (G.MOK). Example 11: Demonstration of the immunogenic power of the glycoprotein of the Mokola virus (G. MOK).
Des cellules de Spodoptera frugiperda (SF9), sensibles au baculovirus, sont infectées avec un baculovirus exprimant la G.MOK ou avec un baculovirus sauvage n'exprimant pas le gène de la polyédrine (NPEV del). Trois jours après l'infection, ces cellules sont lavées en tampon PBS. Différentes dilutions de ces cellules sont alors réalisées pour tester la valeur protectrice de cette suspension cellulaire chez des souris par un test de HABEL modifié. Des lots de 10 souris sont vaccinés par voie intrapéritonéale, deux fois à une semaine d'intervalle et éprouvés par inoculation intracérébrale, avec différentes dilutions de virus, deux semaines aprè la première vaccination. Ces souris sont alors observée quatre semaines après l'épreuve. A l'issue de ce délai, un pourcentage de mortalité spécifique est calculé.
La figure 9 montre que les courbes de mortalité de souris non vaccinées (NV) et vaccinées avec des cellules SF9 infectées avec NPEV del sont peu différentes. Les cellules SF9 infectées par NPEV del n'entraînent donc aucune protection non spécifique des souris. Cells of Spodoptera frugiperda (SF9), sensitive to baculovirus, are infected with a baculovirus expressing G.MOK or with a wild baculovirus not expressing the polyhedrin gene (NPEV del). Three days after infection, these cells are washed in PBS buffer. Different dilutions of these cells are then made to test the protective value of this cell suspension in mice by a modified HABEL test. Batches of 10 mice are vaccinated intraperitoneally, twice at one week intervals and tested by intracerebral inoculation, with different dilutions of virus, two weeks after the first vaccination. These mice are then observed four weeks after the test. At the end of this period, a specific percentage of mortality is calculated. FIG. 9 shows that the mortality curves of unvaccinated (NV) mice and vaccinated with SF9 cells infected with NPEV del are little different. SF9 cells infected with NPEV del therefore do not give any non-specific protection to mice.
La figure 10 montre qu'une dose vaccinale de 100 000 cellules SF9-G.MOK suffit pour protéger des souris et 106 cellules SF9-G.MOK suffisent pour obtenir un index de protection supérieur à 2,5. FIG. 10 shows that a vaccine dose of 100,000 SF9-G.MOK cells is sufficient to protect mice and 10 6 SF9-G.MOK cells are sufficient to obtain a protection index greater than 2.5.
Ainsi que cela ressort de ce qui précède, l'invention ne se limite nullement à ceux de ses modes de mise en oeuvre, de réalisation et d'application qui viennent d'être décrits de façon plus explicite ; elle en embrasse au contraire toutes les variantes qui peuvent venir à l'esprit du technicien en la matière, sans s'écarter du cadre, ni de la portée, de la présente invention.
As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.
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Claims
(1) un fragment de génome d'un baculovirus, comportant la région de contrôle en 5' du gène de la polyédrine ; (1) a genome fragment of a baculovirus, comprising the 5' control region of the polyhedrin gene;
(2) un polylinker approprié à l'insertion d'un gène ou d'au moins un fragment de gène du virus Mokola ; (2) a polylinker suitable for the insertion of a gene or at least one gene fragment of the Mokola virus;
(3) les séquences de contrôle en 3' avec notamment le site de polyadénylation de la polyédrine, ledit vecteur navette permettant la multiplication de la construction. (3) the 3' control sequences with in particular the polyadenylation site of polyhedrin, said shuttle vector allowing the multiplication of the construction.
41°) Vecteur d'expression selon l'une quelconque des revendications 39 et 40, caractérisé en ce que le gène ou un fragment du gène de la glycoprotéine G du virus Mokola est inséré au niveau du polylinker, de manière à obtenir un vecteur d'expression dit chargé de ladite glycoprotéine, sous contrôle du promoteur du gène de la polyédrine. 41°) Expression vector according to any one of claims 39 and 40, characterized in that the gene or a fragment of the Mokola virus glycoprotein G gene is inserted at the polylinker, so as to obtain a vector d the expression said to be loaded with said glycoprotein, under the control of the promoter of the polyhedrin gene.
42°) Vecteur d'expression selon la revendication 41, caractérisé en ce qu'il a été déposé sous le numéro 1-851 en date du 22 mars 1989 auprès de la Collection Nationale de Cultures de micro-organismes tenue par l'Institut Pasteur. 42°) Expression vector according to claim 41, characterized in that it was deposited under number 1-851 on March 22, 1989 with the National Collection of Microorganism Cultures held by the Pasteur Institute .
43°) Vecteur d'expression selon l'une quelconque des revendications 39 et 40, caractérisé en ce que le gène ou un fragment du gène de la nucléoprotéine N est inséré au niveau du polylinker, de manière à obtenir un vecteur d'expression dit chargé de ladite nucléoprotéine, sous contrôle du promoteur du gène de la polyédrine. 43°) Expression vector according to any one of claims 39 and 40, characterized in that the gene or a fragment of the N nucleoprotein gene is inserted at the polylinker, so as to obtain a so-called expression vector loaded with said nucleoprotein, under the control of the polyhedrin gene promoter.
44°) Vaccin contre le virus Mokola, à usage humain et/ou vétérinaire, caractérisé en ce qu'il comprend au moins un peptide et/ou un fragment de peptide
selon l'une quelconque des revendications 31 à 37, éventuellement associé à au moins un véhicule pharmaceutiquement acceptable. 44°) Vaccine against the Mokola virus, for human and/or veterinary use, characterized in that it comprises at least one peptide and/or a peptide fragment according to any one of claims 31 to 37, optionally associated with at least one pharmaceutically acceptable vehicle.
45°) Vaccin selon la revendication 44, caractérisé en ce qu'il comprend la glycoprotéine G et/ou un fragment de celle-ci et/ou la nucléoprotéine N ou un fragment de celle-ci. 45°) Vaccine according to claim 44, characterized in that it comprises glycoprotein G and/or a fragment thereof and/or nucleoprotein N or a fragment thereof.
46°) Vaccin polyvalent des Lyssavirus, caractérisé en ce qu'il comprend au moins un peptide et/ou fragment peptidique selon l'une quelconque des revendications 31 à 37, éventuellement associé à au moins un peptide et/ou un fragment peptidique d'au moins un autre serotype de Lyssavirus. 46°) Polyvalent Lyssavirus vaccine, characterized in that it comprises at least one peptide and/or peptide fragment according to any one of claims 31 to 37, optionally associated with at least one peptide and/or a peptide fragment of at least one other Lyssavirus serotype.
47°) Vaccin selon l'une quelconque des revendications 44 à 46, caractérisé en ce que lesdits peptides et/ou fragments peptidiques sont avantageusement associés à un support approprié et/ou un adjuvant acceptable, notamment les adjuvants classiques des vaccins humains et vétérinaires. 47°) Vaccine according to any one of claims 44 to 46, characterized in that said peptides and/or peptide fragments are advantageously associated with an appropriate support and/or an acceptable adjuvant, in particular the conventional adjuvants of human and veterinary vaccines.
48°) Anticorps monoclonaux spécifiques des peptides et/ou fragments peptidiques du virus Mokola, caractérisés en ce qu'ils résultent de l'immunisation de mammifères, notamment de rongeurs, et plus particulièrement de souris, par des peptides et/ou fragments peptidigues selon l'une quelconque des revendications 31 à 37. 48°) Monoclonal antibodies specific for peptides and/or peptide fragments of the Mokola virus, characterized in that they result from the immunization of mammals, in particular rodents, and more particularly mice, with peptides and/or peptide fragments according to any one of claims 31 to 37.
49°) Procédé immunologique de détection d'anticorps anti-peptides et/ou fragments peptidiques du virus Mokola, qui consiste à détecter les anticorps anti- Mokola éventuellement présents dans un échantillon biologique à l'aide d'un peptide ou d'un fragment de peptide selon l'une quelconque des revendications 31 à 37, en mettant en présence ledit échantillon biologique avec ledit/lesdits peptides ou fragment (s) de peptide, auxquels se lient les anticorps anti-Mokola si de tels anticorps sont présents dans l'échantillon biologique à
analyser, la lecture du résultat étant révélée par un moyen approprié notamment EIA, RIA, fluorescence. 49°) Immunological method for detecting anti-peptide antibodies and/or peptide fragments of the Mokola virus, which consists of detecting anti-Mokola antibodies possibly present in a biological sample using a peptide or fragment peptide according to any one of claims 31 to 37, by bringing said biological sample into contact with said peptide(s) or peptide fragment(s), to which the anti-Mokola antibodies bind if such antibodies are present in the biological sample to analyze, the reading of the result being revealed by an appropriate means in particular EIA, RIA, fluorescence.
50°) Procédé selon la revendication 49, caractérisé en ce que lesdits peptides ou fragments peptidiques sont fixés sur un support solide approprié. 50°) Method according to claim 49, characterized in that said peptides or peptide fragments are fixed on an appropriate solid support.
51°) Procédé selon la revendication 49 ou la revendication 50, caractérisé en ce que lorsque l'on met en oeuvre une méthode de type sandwich, la révélation est réalisée au moyen d'un deuxième anticorps dirigé contre l'anticorps à doser et marqué de manière appropriée. 51°) Method according to claim 49 or claim 50, characterized in that when a sandwich type method is used, the revelation is carried out by means of a second antibody directed against the antibody to be assayed and marked appropriately.
52°) Procédé de détection rapide et spécifique du virus Mokola qui consiste à détecter un virus Mokola, éventuellement présent dans un échantillon biologique à l'aide d'au moins une sonde nucleotidique selon la revendication 29 ou la revendication 30, en mettant en présence ledit échantillon biologique traité de manière appropriée avec ladite/lesdites sondes nucléotidiques, à laquelle/auxquelles se lie l'ARN genomique et/ou les produits de transcription du virus Mokola, si de tels produits sont présents dans l'échantillon, la lecture du résultat étant révélée par un moyen approprié. 52°) Method for rapid and specific detection of the Mokola virus which consists of detecting a Mokola virus, possibly present in a biological sample using at least one nucleotide probe according to claim 29 or claim 30, by bringing together said biological sample appropriately treated with said nucleotide probe(s), to which genomic RNA and/or Mokola virus transcription products bind, if such products are present in the sample, reading the result being revealed by an appropriate means.
53°) Kit prêt à l'emploi, pour la mise en oeuvre du procédé de détection et/ou d'identification d'au moins un Lyssavirus selon l'une quelconque des revendications 1 à 12, caractérisé en ce qu'il comprend outre des quantités utiles de tampons et de réactifs appropriés pour la mise en oeuvre de ladite détection, des doses appropriées d'au moins deux amorces convenables, des doses appropriées d'au moins une sonde nucleotidique et des doses appropriées d'au moins une enzyme de restriction. 53°) Ready-to-use kit, for implementing the method of detection and/or identification of at least one Lyssavirus according to any one of claims 1 to 12, characterized in that it further comprises useful quantities of buffers and reagents suitable for carrying out said detection, appropriate doses of at least two suitable primers, appropriate doses of at least one nucleotide probe and appropriate doses of at least one enzyme of restriction.
54°) Kit prêt à l'emploi pour la mise en oeuvre du procédé de détermination dans un échantillon biologique, d'anticorps anti-Mokola selon l'une quelconque des revendications 49 à 51, caractérisé en ce qu'il comprend au moins :
- des doses appropriées d'au moins un peptide et/ou un fragment peptidique selon l'une quelconque des revendications 31 à 37 ; et 54°) Ready-to-use kit for implementing the method for determining, in a biological sample, anti-Mokola antibodies according to any one of claims 49 to 51, characterized in that it comprises at least: - appropriate doses of at least one peptide and/or a peptide fragment according to any one of claims 31 to 37; And
- des quantités utiles de tampons appropriés pour la mise en oeuvre de ladite détection. - useful quantities of buffers suitable for carrying out said detection.
55°) Kit selon la revendication 54, caractérisé en ce que le peptide est de la glycoprotéine G selon la revendication 35 ou la revendication 37 ou un fragment de celle-ci, fixée sur un support solide approprié. 55°) Kit according to claim 54, characterized in that the peptide is glycoprotein G according to claim 35 or claim 37 or a fragment thereof, fixed on an appropriate solid support.
56°) Kit selon la revendication 54, caractérisé en ce que le peptide est de la nucléoprotéine N selon la revendication 32 ou la revendication 37 ou un fragment de celle-ci, fixée sur un support solide approprié. 56°) Kit according to claim 54, characterized in that the peptide is the N nucleoprotein according to claim 32 or claim 37 or a fragment thereof, fixed on an appropriate solid support.
57°) Kit prêt à l'emploi pour la mise en oeuvre du procédé de détection d'un virus Mokola selon la revendication 52, caractérisé en ce qu'il comprend au moins : 57°) Ready-to-use kit for implementing the method for detecting a Mokola virus according to claim 52, characterized in that it comprises at least:
- des doses appropriées d'au moins une sonde et/ou fragment de sonde nucleotidique selon la revendication 29 ou la revendication 30 ; et - appropriate doses of at least one probe and/or nucleotide probe fragment according to claim 29 or claim 30; And
- des quantités utiles de tampons appropriés pour la mise en oeuvre de ladite détection.
- useful quantities of buffers suitable for carrying out said detection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR898904052A FR2645173B1 (en) | 1989-03-29 | 1989-03-29 | CLONING AND EXPRESSION OF GENES ENCODING PEPTIDES AND / OR FRAGMENTS OF MOKOLA VIRUS PEPTIDES, APPLICATION TO THE PREPARATION OF A V |
| FR8904052 | 1989-03-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0465602A1 true EP0465602A1 (en) | 1992-01-15 |
Family
ID=9380140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP90907112A Withdrawn EP0465602A1 (en) | 1989-03-29 | 1990-03-29 | METHOD FOR DETECTION AND/OR IDENTIFICATION OF $i(LYSSAVIRUS) INFECTIONS, CLONING AND EXPRESSION OF GENES CODING FOR PEPTIDES AND/OR FRAGMENTS OF PEPTIDES OF MOKOLA $i(LYSSAVIRUS), VACCINE AGAINST THE MOKOLA VIRUS AND/OR THE FAMILY OF $i(LYSSAVIRUSES) AS WELL AS METHOD FOR OBTAINING SAID VACCINE VIA |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0465602A1 (en) |
| JP (1) | JPH04506747A (en) |
| FR (1) | FR2645173B1 (en) |
| OA (1) | OA09556A (en) |
| WO (1) | WO1990011358A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992020820A1 (en) * | 1991-05-15 | 1992-11-26 | Vanderbilt University | Method to determine metastatic potential of tumor cells |
| CN114957458B (en) * | 2021-05-08 | 2023-10-31 | 南昌大学 | High-affinity anti-rabies virus fully human monoclonal antibody and application thereof |
| CN114989296B (en) * | 2022-06-21 | 2023-08-11 | 龙湖现代免疫实验室 | Monoclonal antibody 2F2 of rabies virus and general type rabies virus antibody rapid detection test paper |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU606043B2 (en) * | 1985-03-28 | 1991-01-31 | F. Hoffmann-La Roche Ag | Detection of viruses by amplification and hybridization |
| AU599348B2 (en) * | 1985-12-18 | 1990-07-19 | Microgenesys, Inc. | Method for producing selected polypeptides in virally infected insect cells and polypeptides isolated therefrom |
| EP0237686A1 (en) * | 1986-03-18 | 1987-09-23 | Institut Pasteur | DNA sequences derived from the rabies virus genome |
| CA1337115C (en) * | 1987-07-30 | 1995-09-26 | Bernhard Dietzschold | Vaccination against rabies-related diseases |
-
1989
- 1989-03-29 FR FR898904052A patent/FR2645173B1/en not_active Expired - Fee Related
-
1990
- 1990-03-29 JP JP2506608A patent/JPH04506747A/en active Pending
- 1990-03-29 WO PCT/FR1990/000216 patent/WO1990011358A1/en not_active Application Discontinuation
- 1990-03-29 EP EP90907112A patent/EP0465602A1/en not_active Withdrawn
-
1991
- 1991-09-27 OA OA60078A patent/OA09556A/en unknown
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9011358A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2645173B1 (en) | 1994-06-17 |
| FR2645173A1 (en) | 1990-10-05 |
| OA09556A (en) | 1993-01-31 |
| WO1990011358A1 (en) | 1990-10-04 |
| JPH04506747A (en) | 1992-11-26 |
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