JPH04506747A - Methods for detection and/or identification of lyssavirus infections, cloning and expression of genes encoding peptides and/or fragments of peptides of Mokora lyssavirus, vaccines against Mokora virus and/or the lyssavirus group, and methods for the aforementioned by genetic engineering. Vaccine manufacturing method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for detecting and/or identifying lyssavirus infection, Mokolara lyssavirus bev Cloning and expression of genes encoding fragments of peptides and/or peptides; Vaccines and genetic engineering against the coravirus and/or lyssavirus group Method for producing the vaccine according to science This invention provides a method for detecting and identifying Lyssavirus infection; Bebutide of Mokola Lyssavirus and Cloning and expression of genes encoding peptide fragments, Mokola virus and/or vaccines against the lyssavirus group and genetic engineering. Regarding the production method of cutin.

Rhabdovirus, a virus related to rabies, has been spreading for the past 20 years. virus), whose classification has been confirmed based on the cross serum neutralization method and complement fixation method. However, it has been isolated all over the world. Thus, the genus Lyssavirus is associated with rabies virus ( serotype 1), Lagos bat virus (serotype 2, Africa), Mokola virus (serotype 3, Africa) and Duebenhag virus (serotype 4, South Africa) The serotypes were separated into four different serotypes, each represented by a. northern europe Viral strains of lyssavirus isolated from bats in Spain and Spain are currently being classified. be. These viruses have currently been isolated only from bats, but they have also been isolated from humans, etc. The issue of conservation of animal species, in particular the issue of vaccine protection against these viruses. is raising the issue. Mokolavirus has spread to many species in Africa, including humans. Isolated cases of fatal rabies encephalitis in Zimbabwe, particularly meat consumption involving dogs The cause of epidemics that occur even if domestic animals are vaccinated against rabies. This is particularly important because it has been

For more information on the nucleotide sequence of the rabies virus and the vaccines derived from it, It has been described in numerous publications.

'yia, 9=7n stay f (WiSTARrNsTrTUTE) (7) 7 -7 The patent encodes a glycoprotein of the ERA strain of the rabies virus, and defined by its nucleotide sequence, its start codon ATG and its stop codon TCA is a synthetic single-stranded RNA that is a copy of the mRNA of the glycoprotein. It presents complementary DNA.

The polypeptide fragment of the cDNA between the two cleavage sites is 50 or more amino acids. It never happens.

The functional amino acid sequence of this glycoprotein precedes the amino-terminal lysine residue and A signal peptide composed of I9 nonpolar amino acids that is cleaved into mature proteins. Contains 524 amino acids that promote growth.

Its molecular weight is approximately 67,000.

Rabies virus cDNA and glycoprotein mRNA are used to carry out immunization. The method used to transform bacteria to produce polypeptides in sufficient quantities. I can do it.

TRANSGENE's European patent application no. Vectors such as phages or plasmids that express proteins, especially glycoproteins - these vectors contain at least one protein encoding said protein. containing a functional DNA sequence and a promoter for the expression of said sequence in bacteria, The effective DNA sequence that it encodes is the entire sequence contained between the two defined cleavage sites. It can be a functional DNA sequence or a subsequence.

This vector depends on whether it is a plasmid or a phage. , used to transform or transfect bacteria, and to culture the bacteria. The desired rabies antigen protein is obtained by It is defined by its amino acid sequence and is used as the active ingredient in anti-rabies vaccines.

European Patent Application No. 237, 686 by Anstitu Pasteur and CNR5 ( March 18, 1986) is Nucleic Ac1ds Res, Volume 14, 6, pp. 2671-2683, 1986 and Proc, Natl, Acad, S. ci, USA, Vol. 83, pp. 3914-3918, 1986. The data is reproduced, but the rabies virus is unsegmented (unsegmented). nted) presents a partial sequence (up to position 5500) of a single unmodified ff1 RNA. However, this sequence is similar to the cDNA derived from the above RNA. It is.

However, the scope of the claims of this application mainly lies in the partial portions encoding nuclear proteins. Rinucleotide sequence 71-1421. Partial sequence 15 encoding Ml protein 14-2404, partial sequence 2496-3102 encoding M2 protein, and and partial sequence 5417-5500 encoding the N-terminal protein of L protein Regarding.

In addition, when these nucleotide sequences and their fragments are inserted into a vector, It is suggested in this application that these vectors be modified appropriately. Once introduced into a host cell, these vectors transfer the DNA sequence into the host cell. The protein is copied and translated to produce the corresponding protein, and then the protein is transferred to the host. It can be isolated from cell extracts of principal cells.

This application also claims that the We are dealing with recombinant DNA containing one of the above-mentioned inserts. This DN A is the transformation of eukaryotic cells (e.g. Vero cells) to produce proteins with immunological properties. vectors that can be used to produce Viral or eukaryotic promoters recognized by the polymerase of the cell Furthermore, the above DNA'A has an appropriate polypeptide downstream of the insert. Contains an adenylation site. The patent application further states that the main advantages of the invention are: Rabies virus that can be isolated in a form that lacks the nucleoprotein rather than the genome itself The purpose of this study is to provide DNA sequences derived from the genomic RNA of . The cDNA of this patent application can be used to detect rabies viruses in biological fluids, for example by hybridization. It can be used as a probe to detect the presence of viruses.

As defined by the peptide structure in the above patent application and the above literature, The polypeptides N5M1 and M2 themselves contain viral polypeptides in biological fluids. The M1 protein can be used to diagnose in vitro the presence of Very specific as an immunogenic composition in combination with excipients used in the manufacture of vaccines. shows significant advantages.

N., TORDO et al., Virol, vol. 165, pp. 565-576, 1988 In 2013, he described a method for determining the complete sequence of the rabies virus genome. They are divided in the viral protein (polymer) that contains unmodified native RNA. , found a highly protected domain.

0, POCH et al., Biochemie, 70412.1019-102 p. 9, 1988, c of the genome of an avirulent strain of rabies virus (A'101). Describes DNA fragments. The sequence of 3386 nucleotides from the 3' end is Not only the intergenic region but also the leader sequence RNA5N nuclear protein, M1 phosphorus protein spans genes encoding protein and M2 matrix proteins . Comparison of the AVO1 sequence with the sequences of other strains of rabies virus revealed that the nucleotide A high degree of conservation has been revealed in both the amino acid and amino acid levels.

The rabies virus genome was synthesized with non-segmented negative single-stranded RNA [rhabdovirus and Other viruses containing paramyxovirus When compared to the genome of phosphoproteins and matrices that are localized at the ends of each gene and are involved in the transcription process. The regions of the protein maintain the same overall structure.

The results presented in this article demonstrate that the unique features of rabies virus transcription are distinct and This suggests that it is in a fluctuating genetically open region.

Mokola virus is a so-called “related” rabies virus, and the rabies vaccine is It provides only weak protection against infections caused by Mokola virus.

Vaccination failures were investigated in the laboratory. Three types of commercially available vaccines (PV strain (Pass2) Prevention of Flury strain (Behring), PV strain (Merieux); Flury strain (Behring) Inoculated mice were non-resistant to Mokola virus infection. the result, These sera contain small amounts of antibodies that can neutralize Mokola virus in vitro. These lymphocytes do not contain any molecules, and these lymphocytes are able to reach target cells infected with Mokola virus. shows low cytotoxicity against

Develop, Biol, 5tand, by T. J. Wiktor et al. Volume 57, pages 199-211, 1984, using monoclonal antibodies, An analysis of the antigenicity of rabies virus and mokola virus is described, and rabies virus Unlike other related viruses, Mokola virus cannot be vaccinated against rabies. It is clearly stated that serum from individuals who have been exposed to the virus does not neutralize the virus.

E, CeLi5 et al. J, Viro1. ．． Volume 3, pages 3128-3134, 1 In 988, humans were immunized with peripheral blood mononuclear cells and PM anti-rabies vaccine. The T clone of an individual is infected with antigenic determinants of rabies virus and rabies-related viruses. The recognition properties were tested in the antigen-induced proliferation assay (A[PA). T Some, if not all, of the cells contain various experimental strains of rabies virus and Crossing over with related viruses like Duvenhage and Mokola Show reaction. These T cells contain viral glycoproteins or liponucleoproteins. Reacts with epitopes of quality.

These publications cover viruses related to rabies virus, such as Mokola virus. Method for producing genomic cDNA clones, or prophylactic, therapeutic, or diagnostic agents thereof There is no mention of its use as such.

Furthermore, some of these documents refer to rabies virus and rabies virus related viruses. There is cross-reactivity between the anti-rabies vaccine and the Mokola virus. This indicates that it does not protect against related viruses such as viruses.

Therefore, groups at risk of infection (veterinarians, animal caretakers, laboratory personnel) and livestock to prevent infection of humans and to treat individuals after exposure to the risk of infection. It is necessary to provide vaccines against the above-mentioned viruses for use in animals and/or animals. That is clear.

Attenuated vaccines have a virus titer of the order of 10 to 10' obtained in cell culture. It appears that it is currently not manufactured on an industrial scale due to its low temperature.

Therefore, the production of anti-Mokola vaccines by genetic engineering offers great benefits It is something.

As far as rabies diagnosis is concerned, the presence of rabies virus in the suspected specimen must be confirmed. Two methods are currently being used to clarify this.

*Immunofluorescence (IF) or immunoenzymatic testing (RREID) How to use antibodies to study dog disease antigens * Inoculate mice with virus Methods for detecting viruses in cell culture (CC) are specifically mentioned. Ru.

A group of monoclonal antibodies that can identify different strains of rabies are now available. It is Noh. Some of these monoclonal antibodies belong to the genus Lyssavirus. specific for several serotypes and especially for isolates belonging to the same serotypes . These monoclonal antibodies therefore provide accurate characterization of various lyssavirus strains. decisions can be made and can be used in epidemiological studies.

However, the identification of bacterial strains that is possible with this method generally requires culturing these strains in advance. It takes a long time because it is necessary. Each new isolate undergoes fusion to create a new It is necessary to create highly specific monoclonal antibodies.

A, Mo1. of Erm1ne et al. Ce11. Probes, Volume 2, 75-8 Page 2, in 1988, a rapid diagnosis method for rabies infection using the hybridization method was described. '%sru.

This hybridization method is used to detect rabies transcripts in the brain. P-crazy A cDNA probe labeled with 21p derived from the genome of a canine disease strain (serotype l) was Used to immobilize very small amounts of specific viral RNA. Purified virus R NA is obtained after phenol extraction. The RNA was immobilized on a nylon membrane, and each rabid dog M13 insertion complementary to disease gene and 200-400 nucleotides of each mRNA Pool the fragments and hybridize. Autoradiolog the hybridized labeled probe. Detect with roughy. Hybridization is caused by the rabies virus Vulpine. ) strain (serotype l). 80ng A positive reaction was obtained for the amount of total RNA. Detection of viral transcripts in animals This was also possible for brain samples collected one week after death. fluorescent anti-rabies Isolating rabies virus using antibodies or on mouse neuroblastoma cells Complete correlation compared to other methods such as detecting rabies antigens by has been observed.

However, these methods, on the one hand, use saliva or organ samples to Mokola virus infection cannot be detected rapidly, especially in biological samples. cannot be directly detected or identified.

Negative Ippon held in Dinard, France from September 18th to 23rd, 1988. At the 7th International Conference on Chain Viruses, the inventors announced that by genetic engineering , the cloning of Mokola virus with the aim of making it possible to produce a vaccine. provided an informational text describing their research on This informational summary contains Mokola virus recovered from the supernatant of infected BHK21 cells was purified and It is stated that genomic RNA was extracted from

The purpose of this invention is to express viral antigens primarily associated with immune responses. A specific vaccine against Mokola virus obtained by The aim is to provide multivalent vaccines against virus serotypes and Vaccine production requires solving the problems of virus production and preventing lyssavirus infections. This has the advantage of providing a specific and highly sensitive diagnostic agent.

Another object of this invention is to rapidly detect lyssavirus infections from saliva or organ samples. The objective is to provide a method for detecting

The goal of this invention is to rapidly detect small amounts of lyssavirus present in biological samples. and/or a method of identifying a lyssavirus, wherein said sample is optionally present. To extract the viral RNA and/or transcripts of This method is characterized by: That is, (1) Lysviruses to obtain cDNA from genomic RNA or mRNA contacting with at least one suitable primer for (2) Then, in order to amplify at least one fragment of the above cDNA sequence, contacting the cDNA sequence with a pair of appropriate primers for Lyssavirus; One of these primers is different from the primer in the above step (]), and the other primer is - is the same as or different from the primer in step (1), (3) then detecting the amplified cDNA sequence by appropriate means. It's a method.

The PCR method is described in particular by 5aiki et al., 5ciense, 239.487.198 8 and cetus European Patent Application No. 200362 and European Patent Application No. 201184 It is described in. However, the method of the present invention surprisingly allows saliva or Rapid detection of lyssavirus infections from organ samples1, (within 12 hours), then The lyssavirus can be identified.

According to an advantageous embodiment of the detection method, the primers are primers specific for the bacterial strain. In the case of Lysvirus serotypes, the Lysvirus serotypes Consists of specific primers and conserved sequences of genes common to all lyssaviruses Primers that are used as primers and are hereinafter referred to as multivalent primers. selected from the group consisting of:

The multivalent primer of this invention itself can be used for diagnosis or treatment of lyssavirus. Primers for detection (lyssavirus infection, yes/no response) and differential diagnosis Primer for cutting or typing [lyssavirus infection, type (typing)] A distinction can be made between

According to an advantageous feature of this embodiment, the primers are of the rabies and mokola genomes. 3' rabies primer localized at positions 1-18.

According to another advantageous feature of this embodiment, the primers are 2901 of the rabies genome. M2 rabies primer localized at position ~2918.

According to another advantageous feature of this embodiment, the primers contain 466 nucleotides of the rabies genome. 5 to 4687 and 23 localized at positions 4675=4697 of the Mokola genome. This is a rabies primer consisting of nucleotides, hereinafter referred to as G primer.

According to another advantageous feature of this embodiment, positions 5520-5543 of the rabies genome and 24 nucleotides located at positions 5545-5568 of the Mokola genome. This is a rabies primer composed of the following rabies primers, and is hereinafter referred to as L primer.

According to yet another advantageous feature of this embodiment, the primer is used for rabies and mokola. Mokolaply consists of 18 nucleotides located at positions 587-605 of the genome. rabies primer, hereinafter referred to as Na primer.

According to a further advantageous feature of this embodiment, the primer is used for rabies and mokola. Moco is composed of 16 nucleotides located at positions 1013-1029 of the genome. Nb primer or rabies primer, hereinafter referred to as Nb primer.

According to an advantageous embodiment of the method, the detection of the amplified nucleotide sequence comprises Hybridization with one probe or groups of probes appropriate for the species of lyssavirus It will be carried out in

Other advantageous implementations of the method described above! 11! ! According to Mr. Detection of the sequence involves cleaving said sequence with at least one group of appropriate restriction enzymes and then This is carried out by separating the separated fragments by electrophoresis.

According to an advantageous feature of this embodiment, the enzyme group is BamHT.

It is composed of Hind II, Hind III, and Pst.

According to another advantageous feature of this embodiment, the enzyme group is Rsa r, Taq It is composed of E, BstX I and Fnu4H [.

Sequence homology between serotypes 1 and 3, which are currently the most divergent among the lyssaviruses. Depending on the assay method, the Conserved or variable regions can be defined that determine each selection.

Selected multivalent primers can amplify conserved regions of lyssaviruses The method of the present invention for rapid detection of small amounts of lyssavirus in a sample In contrast to domestic animals, animals collected from livestock that are suspected of being the source of initial infection or human contamination. The collected saliva samples can be used to diagnose lyssavirus infection, and in practice , for example, this gene is located not too far away (400 bp) from the N gene. A pair of primers, namely a Na primer and a Nb primer, as defined in -, even if the lyssavirus is present in very small quantities and the sample is severely degraded. It is possible to detect lyssavirus (YES/NO response) even when the virus is present. actually, The sensitivity of conventional methods is limited by this rapid onset, where diagnosis can be performed in less than 12 hours. It is lower than the method of Ming.

The selected primers are linked to the variable region of the lyssavirus (e.g. pseudogene Psi). If it is possible to synthesize and amplify a region of the Ml gene, the method of this invention can identify and type the lyssaviruses involved in the infections to be detected. Wear. In fact, a pair of primers as defined above, namely the rabies G primers and Rabies L primers are combined with a panel of appropriate enzymes to target each of the rabies viruses. Type seedling stocks, Mokorovirus, and Lyssvirus strains isolated from bats. Can be separated. One of the above two primers is positioned distal to the G gene. This corresponds to a conserved region of lyssavirus located proximal to the L gene, and this The region was separated by approximately 860 nucleotides in the whole rabies method and Mokola virus. It is chosen to be able to amplify the displacing region (pseudogene Psi). that amplified The bands may be due to hybridization with probes that are more or less specific for the appropriate strain, or Hybridization in the presence of restriction enzymes more or less specific for the strain detected Reotsu.

Direct sequencing methods also apply directly to agarose pieces containing amplified bands. can be done.

Furthermore, the present invention relates to a specific sequence of Mokola virus, and by this sequence, The conserved and variable regions within the genus Lyssavirus can be detected, and the rabies virus Assess the variability of each genomic region by comparing the Nome and Mokola genomes It is possible to develop methods for detecting and/or identifying the above-mentioned lyssaviruses. I can do it. The cDNA nucleotide sequence from Mokola virus genomic RNA is , is characterized by consisting of approximately 12,000 nucleotides.

The cDNA sequence from the genomic RNA further has complementary 3' and 5' ends. The 12 nucleotides at the 5° end are identical to those of the PV strain of the rabies virus. The “leader” sequence RNA is encoded continuously from the 3′ end to the 5° end. genes that encode proteins, and then genes N, Ml, M2, G, and L that encode proteins. It has the characteristic of having

According to an advantageous embodiment of said cDNA sequence from Mokola virus genomic RNA. For example, the cDNA sequence is the following nucleotide sequence and amino acid sequence (1): I have it.

(The following margin continues from the bottom line) This invention provides a pre-encoding method for peptides and/or fragments of Mokolavirus. Fragments of this sequence and the variation zone of the Mokola genome, i.e. Nonconserved zone of genes common to Viridae , and a non-coding fragment of said sequence corresponding to .

Among said code fragments, the sequences are inter alia:

・Nucleotides 71-1420 of the cDNA sequence that encodes the NM protein a fragment corresponding to the cD'NA sequence encoding the M1 protein; Fragment corresponding to nucleotides 1524-2432, encodes M2 protein and the flag corresponding to nucleotides 2516-3121 of the cDNA sequence. nucleotide 332 of the cDNA sequence, encoding the G protein. The fragment corresponding to 8-4893 and the NH-terminus of the L protein, c Sequence corresponding to nucleotides 5443-6831 of the DNA sequence.

For non-coding fragments, the sequence is particularly limited to nucleotide 4 of said sequence. 897-5442, and the fragment corresponds to rabies p. Includes those corresponding to si coagulation genes.

Furthermore, this invention consists of approximately 12,000 nucleotides and is non-segmented and non-polyaryin. Denylation-negative, single-stranded RNA with continuous “lead” from 3′ to 5′. The gene encoding the “dar” sequence RNA, followed by the N nucleoprotein and M1 phosphoprotein. , genes encoding M2 matrix protein, G glycoprotein, and L polymerase protein Mokolawi, which is characterized by its genome always being bound to N-nucleoprotein. rus genome RNA sequence.

Moreover, the present invention provides protein N, Ml, M2 . Coding G and L 5 consecutive monosiltronic fragments, i.e. Corresponds to nucleotides 59-1484 of the sequence of formula I combined with polyA fragment, - Combines with poly8 and corresponds to nucleotides 1495-2489 of the sequence of formula I fragment, - Combines with polyA and corresponds to nucleotides 2501-3283 of the sequence of formula ■ fragment, - Combines with poly8 and corresponds to nucleotides 3307-5380 of the sequence of formula ■ Fragment encoding ShiG protein, large fragment encoding L protein , as well as the bicistronic fragment Ml-M2, The present invention relates to a Mokola virus transcript characterized by comprising:

Furthermore, this invention covers the entire genome from the 3' end to the 5' end, i.e. ・Measure 4150 nucleotides (hereinafter referred to as pMD10) and identify the “leader” sequence. sequence RNA, sequences encoding N nucleoprotein, M1 protein, M2 protein and G protein. fragment corresponding to the fragment of ・2850 nucleotides (hereinafter referred to as pMAlo) were measured and encoded G protein. corresponding to the fragment of the sequence that encodes the L protein and the fragment that encodes the L protein. Fragment, consisting of 6830 nucleotides at the 3' end, leader sequence protein N, Ml, M2 and G as well as the first 1420 nucleotides of the L gene. BgII[part] containing the sequence encoding Otide (hereinafter referred to as pM7). A fragment obtained by ligating the insert pieces pMD10 and pMAlo within the ・It has approximately 3300 nucleotides (referred to as pMB5) and encodes L protein. Fragment corresponding to the fragment of (referred to as 9M12), which corresponds to a fragment of the sequence encoding the L protein. a fragment of approximately 700 nucleotides (referred to as pMR15a); fragments of protein-coding sequences as well as the untranscribed 5' end of the genome. Corresponding fragments, these fragments, when separate, each specifically binds RNA or cDNA fragments derived from transcription or reproduction of the system. Cultivate the ability to hybridize, cDNH clone from Mokola virus genomic RNA characterized by corresponding to Regarding the

According to this invention, clone pM7 is derived from Anstituvastur ([n5t Itut Pa5teur) Collection Nacional Dou Curci Microorganism (Collection National l　de　Cu1tures de　IJicrooganismes) , deposited on March 22, 1989 as No. 1-847.

According to this invention, clone pMB5 was deposited with the same depository on March 22, 1989. It was deposited as No. 1-848 on the same day.

According to this invention, clone pM12 was deposited with the same depositary institution on March 2, 1989. It was deposited as No. 1-849 on the 2nd.

According to this invention, clone pMR15a was deposited in the same depository in March 1989. It was deposited as No. 1-850 on the 22nd.

Furthermore, the present invention provides a nucleotide sequence or a fragment thereof as defined above. The agent can be treated with radioactive isotopes, suitable enzymes or manufacturers such as fluorochromes. The present invention relates to a nucleotide probe characterized in that it consists of a nucleotide probe labeled with -.

According to this embodiment of the probe, the sequence 4675-5568 or its flag ment, especially fragment 4897-5442 or its complementary strand. It will be done.

Such clones are specific to Mokola virus.

Moreover, the invention provides at least one fragment, part or part thereof as defined above. is a peptide characterized by being encoded by a combination of several fragments. or a fragment thereof.

According to an advantageous embodiment of the invention, the peptide coexists in the N-nucleoprotein gene. is coded and has the amino acid sequence of the following formula (2).

(：ENG) According to another advantageous embodiment, the peptide is encoded by the M1 protein gene and has the following formula: ■ Cultivate the amino acid sequence of.

(::ENG) According to another advantageous embodiment, the peptide is encoded by the M2 protein gene and has the following formula: Cultivate the amino acid composition of

and is characterized by the amino acid sequence of formula ■.

(V) According to Ike's preferred embodiment, the peptide is encoded by the L protein gene and contains the following amino acids: It has an acid sequence.

Formula (V[) sequence: m) Formula (V[D sequence: Flash:] In an advantageous embodiment of the peptide and/or fragment thereof according to the invention, are obtained by synthesis.

Furthermore, the invention provides at least one nucleotide sequence as defined above. The present invention relates to a vector characterized in that it contains an element or a part thereof.

Moreover, the present invention provides for at least (1) bacculovirus, a naturally occurring bacterium; (2) the genome or at least the genome of Mokola virus; A suitable shuttle consisting of a polylinker suitable for inserting fragments of Nome Mokola virus peptide characterized by being obtained by corresponding recombination between vectors Expression vectors for combinations of fragments, peptide fragments, and peptide fragments Regarding.

According to an advantageous embodiment of the expression vector according to the invention, the shuttle vector ( 1) Bactovirus genome consisting of the 5゛ control region of the polyhetrin gene. (2) a gene or at least a fragment of a gene of Mokola virus; A polylinker suitable for inserting the fragment and (3) especially a polyhetosine polylinker. The construct consists of a 3° control sequence that nurtures the denylation site. It is possible to proliferate.

Advantageous features of embodiments of the invention include the Mokola virus G glycoprotein gene or The gene fragment is linked to the polyhetrin gene promoter at the level of the polylinker. Under the control of the glycoprotein, an expression vector charged with the glycoprotein is obtained. It will be inserted as shown below.

of at least one peptide, fragment thereof or peptide of Mokola virus. Expression methods for combinations of fragments include recombinant vectors expressing the desired polypeptide. To obtain baculovirus, in the presence of natural baculovirus, a suitable lebido in Butella cells, especially Catelpira-5podoptera frugiperda. Using the expression vector according to this invention.

The choice of recombinant baculovirus will depend on the ecology of the resulting plaque. Poly The presence of hetrin can be detected using a light microscope when illuminated with evil luminescence. Gives a refracted appearance to the type of Willis Plague. On the contrary, recombinant virus Sprague's dull appearance is due to the absence of crystals, making them easy to distinguish. I can do it. This representation system has many advantages. That is, Baculovirus can easily undergo glycosylation, phosphorylation, palmitylation, etc.

This allows many proteins to undergo post-translational modification, such as in the case of G-glycoproteins. It becomes possible to devise expressions.

The expression level is extremely high, on the order of 1 to 1 O per lysotol of culture.

It is possible to culture Bydophtera cells in suspension.

-Polyhetrin is expressed after viral replication and nucleocapsid formation The fact that it is a late gene that can express toxic proteins for virus proliferation. become.

Furthermore, the invention provides at least one peptide according to the invention and/or its fragment, optionally in combination with at least one pharmaceutically acceptable excipient. Regarding a vaccine against Mokola virus for humans and/or livestock, characterized by do.

According to an advantageous embodiment of this vaccine, the vaccine comprises G-glycoprotein and/or its constituents. and/or Ni protein or a fragment thereof.

The combination of these two viral peptides mediates various levels of the immune response. . In fact, the G-glycoprotein preferentially induces the formation of neutralizing antibodies, whereas the N-nucleoprotein is particularly Mediates cell-mediated immunity.

The invention also provides at least one peptide according to the invention and/or its fragments. and optionally at least one of at least one other serotype. a peptide and/or a fragment thereof. Regarding virus multivalent vaccine.

Serotype 4 Lysvirus rabies virus), Serum ¥2 Lysvirus (Lagos Komo serotype 4 Lysvirus (Lagos Bat virus), serotype 4 Lysvirus Regarding Duvenhage virus) and serotype 4 A particular example is the lyssavirus isolated from the European bat.

According to the invention, the peptide and/or its fragments are prepared in a suitable excipient and / or in combination with conventional vaccines, especially human and livestock vaccines, with acceptable adjuvants It is advantageous to do so.

This invention relates to mammals, particularly mice, rather than rodents. Mokola virus peptide obtained by immunization with peptide and/or its fragments. The present invention relates to monoclonal antibodies specific for tide and/or fragments thereof.

This invention provides a method for contacting biological samples with peptides or fragments thereof according to the invention. If there is an antibody present in the body, the anti-Mokola antibody will bind, and the result will be displayed. Appropriate means, especially EIA.

Read with RIA or fluorescence to detect vine anti-Mokola antibodies present in biological samples. Anti-Mocola virus bebutide and/or peptide fragment consisting of Regarding immunological methods for detecting the body.

In an advantageous embodiment of this method, the peptide or fragment thereof is placed in a suitable solid state. Immobilized in excipients.

The immunization method may be of the direct or indirect type and may be of the sandwich or vegetable type. You may also use the do method.

Nan: ’6y Chihofei 1.・In other advantageous embodiments, labeled as appropriate, Necropsy is performed with a second antibody to the antibody being assayed.

This method allows serum antibodies to be titrated and serovariants of vaccinated individuals. This allows for the evaluation of conversion or to perform serological probing for immunological purposes.

The present invention also provides the method of preparing at least one generally processed biological sample according to the present invention. contact with a nucleotide probe to detect Mokola virus genomic RNA in the sample. and/or the transcript, if present, binds to the probe and the results are read by appropriate means. , a rapid and specific method for detecting Mokola virus, which consists of detecting Mokola virus in living organisms. Concerning different detection methods.

The invention also provides suitable and useful amounts of buffers and reagents for carrying out the detection, suitable small amounts of at least two suitable primers, an appropriate amount of at least one probe and a suitable at least one restriction enzyme comprising an amount of at least one restriction enzyme. Concerning a simple kit for carrying out the method of the present invention for detecting and/or identifying saviruses. do.

The invention also provides at least one peptide according to the invention and / or an appropriate amount of the fragment, and a useful amount of a suitable buffer to carry out the detection. The present invention relates to a simple kit for carrying out a method for measuring anti-Mokola antibodies.

In an advantageous embodiment of this kit, the peptide is immobilized in a suitable solid excipient. A nuclear protein or a fragment thereof.

This kit contains enzyme, fluorescent or radioisotope-labeled An appropriate amount of a second antibody to the antibody may be added.

The invention further provides at least one blower according to the invention. and/or a useful amount of a suitable buffer for detection. In order to carry out a method for detecting Mokola virus according to the present invention, which is characterized by Regarding the simple kit.

In addition to the above features, other features will become clear from the following description using examples. Would. However, the examples are intended to illustrate the subject matter and are It is not intended to be limited by this.

Example 1 Cloning of DNA complementary to Mokola virus genome RNA and sequencing a) Virus culture The Mokola virus strain studied was collected from a cat native to Zimbabwe.

The viral particles are purified from the resulting plaques on CER cell cultures.

The Mokola virus selected in this way was cultured on BHK-21 cells, and WIKTO The method of R et al. (J. Vivol, 1977, Vol. 21, pp. 626-635) Purify according to a modified method.

b) Purification of genomic RNA In order to isolate the genomic RNA of Mokola virus, the above ff1M virus particles were 1.5% SDS, I00mMh Li-HCl, pH 7,5,100mMNa with C1 and Brotinase K (Lerck) in 10mM EDTA , incubate at 37°C for 30 minutes. followed by phenol chloroform (v ol/vol) twice and precipitated with ethanol.

C) cDNA clone expressing the gene and 5' end of the Mokola virus genome. Characterization and separation 1. Synthesis of CcDNA the first cDNA strand using a 10-fold higher molar ratio. In the presence of 18 g of genomic RNA, 18-mer primers (e.g. rabies 3' primer localized to positions 1-18 of the genome or localized to positions 2901-2918 M2 primer present) and 50 units of reverse transcriptase and 50 mM of Liss hydrochloric acid (pH 8,3), 8mM magnesium chloride, 100mM potassium chloride m, 0.8mM dATP, 0.8mM dGTp, 0.3m M dCTPlo, 3 m M dTTP, 0.2 μM “PdCTP, 0.2 with a buffer containing μM 32PdTTP and 30U/μi’ of RNasin. Synthesis is carried out at 42° C. for 2 hours in the presence of

Double by the method of GUBLER and HOFFMAN C) IOFFVAN) Quantitative cDNA was prepared and separated according to size using a biogel, A-50m column. Ru.

The fragment corresponding to the largest cDNA (approximately 15-20 ng of cDNA) was added to dC/dG In the extension method, insert at the level of the Pst(site) of the PBR322 plasmid.

A competent strain of E. coli (E, Co11) DH5 was transformed and specific Transformants containing the Mokola insert were grown on a nitrocellulose filter. It is detected by forming a hybrid.

2. Characteristics of the obtained clones As shown in Figure 1, start with M2 primer. From the first cDNA library, a rabies probe, namely PV strain (serotype l) Three specific Mokola virus clones were selected, namely 9M B5.9M12, and the characteristics of pMR15a, which spans a 5' portion of the Mokola genome. Perform sex determination.

The cDNA library obtained with the 3-primer 9MA10 clones can be selected with the current Mokola probe. From now on p The MD10 clone is selected.

Inserts pMR15a and pMDlo were sequenced and each It spans the ° end and 3 ° end. This means that the five clones mentioned above are It has been demonstrated that this is sufficient to span the entire genome of the virus.

In Figure 2, the 3' and 5' ends of the Mokola virus genome are shown in the PV strain of the rabies genome. 3” end and 5° end. Complementary nucleotides between these two ends are compared. is represented by a long vertical line.

The beginning of the mRNA for the N gene, the end of the mRNA for the L gene, and the N protein A common sequence that specifies the initiation signal for protein is shown! Ja The 3' and 5' genomic terminal nucleotides of Mokola virus are similar to those of the PV strain. They are identical and are indicated by short vertical lines. The residues are numbered from the end of the genome. be.

Figure 3 shows a southern sample of cells infected with Mokola virus or rabies virus. lot and the non-homologous and homologous rabies probes located on the N and L genes. Hybridization is shown respectively. Obtained from BHK-21 cells Total cytoplasmic RNA was collected after infection with Mokola virus (lane 1) or with rabies. Collected 6.12.24 or 48 hours after virus infection (lane 2) Similarly, it was collected from lane 3, which corresponds to the negative control (cells not infected), and ．． Electrophoresis on a 2% agarose formaldehyde gel. isolated RNA was deposited on a nylon membrane, and the rabies genome cDNA from 377 to 1039 was isolated. sequence (probe NA) or 7034 to 71 of the cDNA of the rabies genome. Hybridization with a 22P-labeled probe consisting of 34 sequences (probe L B) let

Example 2 Characterization of Mokola mRNA synthesized in vivo BHK-21 cells were To determine the in vivo transcript characteristics during coravirus infection, Select cDNA probes according to Figure 1. As shown in Figure 4, six different transcripts observed in Northern plot analysis. Figure 4 also shows the mokola virus game. Mokolawi hybridized with N, N+M, Ml+M2, G and L cDNAs of Nomu. rus mRNA is shown.

Total cytoplasmic RNA of Mokola virus obtained from infected BHK-21 cells was 5. denatured in the presence of 0% formamide, and 15 μg of the sample was denatured in formaldehyde. Electrophoresis is performed on a 1% agar gel containing hydrogen. The isolated RNA was labeled with 22p. Nylon probes (N, M1+M2, N+M1, G and L) of the sampled cDNA. Deposited onto a thin film. Arrows indicate 18S and 2 revealed by staining with ethidium bromide. The position of 8S ribosomal RNA is shown. Various mRNAs are autoradiographed. Fixed fee.

The characterization and size of these Mokola transcripts determine the transcriptional map of Mokola virus. It can be seen that the transcriptional map is the same as that of the rabies virus. In fact, five things Stronic mRNAs appear to begin continuously at the 3' end of the genome. I can do it. Their length is 1750.1250.1000.2400 on agar gel Presumed to be a nucleotide, the tail is polyadenylated.

They encode the N, Ml, M2 and G proteins, respectively. fifth mRNA is estimated to be 4800 nucleotides in length and encodes L protein. However, it is smaller than the expected value due to the length of the genome. The sixth transcript is visible, b icistronic M 1-M 2 c D NA (Line M 1 + Corresponds to M2). Its length is estimated to be 2200 nucleotides.

Example 3 Mokola virus G protein in biological preparations or liquids The invention of anti-G glycoprotein antibody conjugated to horseradish oxidase How to measure in the presence.

The suspension is clarified by centrifugation at 1500×g for 30 minutes at 4°C. centrifugation The clear supernatant of each sample was then coated with an anti-G glycoprotein antibody according to the present invention. Dispense in duplicate into wells of a pre-treated microtiter plate (2 per well). 00μri. The microplate is then incubated for 1 hour at 37°C.

After repeated washing with PBS-Toween buffer, each well was injected with horseradish paste. Add 200 μl of anti-G glycoprotein antibody conjugated to oxidase. Then that ma Incubate the microplate for 1 hour at 37°C and wash again.

Viral antigen can be quantified by the degree of coloring when a peroxidase substrate is added. be done. Its substrate is a mixture of 0-)22-lentiamine and hydrogen peroxide (U). 200μ per well. To accelerate the color reaction, incubate the microplate at room temperature for 2 Leave for 0 minutes. By adding H2SO,N (50μ per well) to stop. Measure the degree of coloration using a spectrophotometer.

Example 4 Conjugation of fluorescein isothiocyanate according to this invention Mokola virus G-glycoprotein was measured in the presence of anti-G-glycoprotein antibody. How to do it.

Cell cultures infected with Mokola virus are fixed in cold acetone for 30 minutes. dyed The color represents the Mokola virus anti-G sugar protein conjugated to fluorescein isothiocyanate. By covering the slide with protein antibody for 30 min at 37°C. Implemented. Evans Blue (115000) is used as a counter dye.

Wash the slides by immersing them in PBS, pH 7.6, for 5 minutes. Group Using a medium containing lyserine, move the slide glass to an Ebfluorescein tube. Test by Roscopy method.

Example 5 Vaccination with peptides and/or peptide fragments according to the present invention Detection method and droplets of Mokola virus anti-G glycoprotein antibody in the blood of seeded individuals Standard method This test consists of a solid phase on which the viral glycoprotein is immobilized, an enzyme conjugate, and a periodine. Staphylococcus aureus C3 taphyloc conjugated with oxidase It is based on the use of protein A of S. occus aureus).

Serum positive and negative control sera are serially diluted 2-fold. Unidentified serum or plasma is 10 Dilute to 0x. Inject 100 μl of each of these serums into wells.

Cover the wells with adhesive film, then place the plate under the thermostat. Incubate for 60 minutes at 37°C in a microplate oven with automatic temperature control. cuvate.

Remove the adhesive film and aspirate the contents of each plate. Wash the wells three times in a row. Clean and dry the plate.

Dispense 100 μl of the above conjugate solution into all wells of all plates, and Cover with film and incubate at 37°C for 60 minutes.

Aspirate the contents of the wells, wash the wells four times, and dry the plate.

Add 100μβ of developing solution (mixture of M pre-solution and peroxidase substrate) to each group. The mixture was added to a well and allowed to react at room temperature for 30 minutes in the dark.

Add 50 μβ of stop solution and read the optical density of each well at 492 nm.

Determine the optical density of the unknown serum as the positive value of the titer expressed as 2 international units per μl. Determine the titer in each well in equivalents per m/m by comparison with a standard curve. can do.

Example 6 Nucleotide probe of the present invention This probe was generated in vector M13. Single strand cloned in genome direction (one direction) or antigenome direction (+ direction) It's about the blobe.

Cloned M2S template 3.5μ! (approximately 0.5 pmol) Rymer 1μ! ! (0.5 pmol) and 0.5 μl of buffer. the The buffer solution was lOmMh Lis HCI, pF (7, 5, 10mM magnesium chloride, Contains 50mM sodium chloride and 1mM DTT. Place the mixture in a tightly closed container. Pour the mixture into a turba, heat it to boiling point, and then slowly boil it on a laboratory bench. Masu. After half an hour, the water reaches 40"C and hybridization is complete.

Add 5 μl of the hybridized template to *2x (4μl! of “p-a-dNTPs)” 4000Ci/mmof, 1mC 1/mf; *Mixture Iμi of 125 picomole/μβ of each of the four dNTPs! : *Make with 1 μl (approximately IU) of E. coli polymerase (Klenow fragment).

The reaction was carried out at 37°C for 20 minutes, followed by heating at 65°C for 3 minutes. It will be stopped.

At this point in the experiment, template M13 has been extracted from the universal primer by the polymerase. determined by the amount of mononucleotide initially introduced into the medium. It becomes double stranded over its length. Furthermore, this length varies statistically from clone to clone. do. Position the probe close enough to the primer to ensure uniform probe size. cleaved at a single restriction site located at the It is two strands.

For this end, mix the synthesis reaction mixture (15 µl) appropriate for the restriction enzyme of choice. (Maniatis et al., 1982), and the cleavage A volume of 20 μl is used for 1 hour at an appropriate temperature.

Then add the same volume of denaturing loading buffer. By heating the mixture After denaturation, transfer onto 6% denatured acrylamide-urea gel (0,5111 [11 thickness)] make a deposit. Continue electrophoresis at 1200 volts for 1 hour. autoradiograph The radioactive probe is located by the fee and then cut out. Over 400 bases In the case of a probe with a size that is not large enough to extract 80% of the radioactivity, Buffer (0,5MNH=C0OH, 10mM MgCILlmM EDTA) It is sufficient to stir the mixture for 1 hour and 30 minutes.

For probes exceeding 400 bases, electroelution )<MANrATIS et al. 1982) is safer. The eluate is Extract with the same volume of saturated phenol, then add ethanol and process as usual. precipitate the tube. After washing twice with 70% ethanol, drying and removing the precipitate Dissolve in 0 μl of water.

4,000 to 10,000 cpm (cerenkof)/μl of labeled probe Obtained by conventional methods.

This probe is then subjected to direct hybridization on the plot or can be used for S1 nuclease mapping. / There are other methods following this. Although it has been slightly modified, it is still the same as usual. The details are described below.

Example 7 Detection of Mokola virus RNA using the probe according to the present invention (RN, A plot) Ingredients below: ・Mixed solution of NaH2PO+ and Na2HPO4 0.5M4:DTA 1 mm ・Hybridization medium containing 1% bovine serum albumin (BSA) prehybridization (up to 2 o'clock rts) and hybridization (generally overnight) and these hybridizations are Both are carried out at 65°C. Wash the filter at the same temperature or with the following buffer: 4 times in a row for 10 minutes each.

・N　a　+　H2P　O4, Na1H+PO<a mixture 40m　M(pH7, 4) ・SD3 1% 4: DTAS pH 7, 51mM To perform hybridization of probes that are strictly complementary to the desired transcript This method gives very good results. Especially when the background is very small. stomach.

Example 8 Method for rapid identification (typing) of small amounts of lyssavirus Stool regular rabies ward, pv, cvs.

AvOL PM, ERA, “Gabon Dog” wild-type rabies virus strain, “Bra B infected with Zircolamori" "European) Ox2, Mokola, etc.) Approximately 1 of total RNA from HK21 fibroblasts or N2A mouse new cells μg is hybridized with approximately 100 μg of “G” primer. This “G” corresponds to the length of 23 nucleotides of the oligonucleotide in the primer (+) direction, It extends between positions 4665 and 4687 of the PV rabies genome; DNA in the complementary (+) direction is specifically primed as described in Example 1. and synthesized. The reaction medium was extracted with phenol and precipitated with ethanol. After that, 50μ of liquid containing the following ingredients! Wash with

L “Primer 1001g Tris-HCI, pH 8,860mM Ammonium sulfate 17mM Mg C1* 6.7mM β-mercaptoethanol tomM EDTA 5μM BSA I70μg/mf DMS0 10% (vol/vol) dNTPs 25mM Taq DNA polymerase 2 units Primer “L” is 24 nucleotides long in the (-) direction of the oligonucleotide. It corresponds to the PV rabies genome and extends from position 552o to 5543 of the PV rabies genome.

Primer ○ is applied to the 4675'-4697 region of the above nucleotide sequence (1). hybridize. Primer G is 4 of Mokola virus sequence (1). Shows approximately 80% homology in the 675-4697 region.

The primers were 5545-5545 of the nucleotide sequence of Mokola virus sequence (1). It specifically hybridizes to position 5568.

The reaction mixture was transferred to a 1.5 mA' capacity Eppendorf tube to prevent evaporation. Add 100 μl of mineral oil from above to create a layer.

and incubate in the "P, C, R, apparatus" according to the following steps: .

Denaturation at +95°C for 5 minutes +50°C for 2 minutes Primer annealing +72°C for 2 minutes M Taq polymer The cDNA chain is extended by the action of lase.

This step is performed once and then repeated 40 times, with a denaturation time of 2 minutes. limited to between

Finally, repeat for the 41st time, but increase the cDNA synthesis time to 10 minutes to complete the reaction. extend in between.

After the reaction product is run on a suitable agar gel, the amplified cDNA band is visualized. can be awakened. After metastasis, the strain already analyzed or another lyssavirus strain The properties can be determined by appropriate probes derived from the cloning of the genome. Wear.

These primers were either immobilized as shown in Figure 7 or wild-type rabies. All available wards were tested serially for Mokola virus. child These primers specifically amplify the pSi pseudogene of any lyssavirus. I can do it. The amplified region of approximately 860 nucleotides has the following characteristics: do.

*Hybridization with radioactive probes that are more or less specific to Lyssavirus strains tion, or hydrolysis with restriction enzymes that are more or less specific for several Type the lyssavirus strains present in a sample within 12 hours without relying on immunology Restriction maps and direct sequencing methods inferred from the region in question for identification.

FIG. 7 shows an electrophoresis gel, which consists of the following 16 lanes.

1. Marker showing size 2-92 Various strains of rabies: namely, rabies virus, PM strain, rabies virus A, VO2 strain, CVS strain of rabies virus, ERA strain of rabies virus, rabies 27 strains of the virus, the Pasteur strain of rabies virus, and the wild-type rabies virus (sheep). ), wild-type rabies virus (sheep) lO: Mokola virus 11-15: Each lining and negative control solution I6・Size marker The amplified cDNA bands were further selected to be able to rapidly distinguish between Hydrolyzed using restriction enzymes.

・Various immobilized rabies wards ・Existing wild-type rabies ward in France ・Mokola virus The following eight restriction enzymes, BamHr, BstXI, Fnu4HI, HindM, Hind TK, PsT I, Rsa L Taq [by doing the following: It is possible to classify the types of various strains.

First, as shown in Table ■, we used a series of four restriction enzymes to and the sources of the various amplified fragments differentiated by the L primers.

As is clear from Table 1, Rsa r qualitatively separates PV from ERA-SAD. However, Taq I quantitatively separates ERA from SAD.

The other two enzymes show stronger group specificity as follows.

・Bstxl only cleaves PV, ERA and SAD ・Fnu4H is wild type Cut only the rabies ward and Mokola virus.

Figure 8 shows that the amplified bands derived from ERA and 073 strains were specifically Bs. It shows that it is cleaved by tX[ and BamH[.

Example 9: Rapid detection method for small amounts of lyssavirus (Yes/N).

response) The primers Na and Nb defined in the present invention are, for example, 587-1029 of the nucleotide sequence of formula (1) of Mokola virus, which is a conserved region @ area can be amplified.

Table I The discrimination is then improved by enzymes such as:

Table■ This method of rapidly detecting small amounts of Norisavirus in a sample according to the present invention also Unlike the current methods, it is possible to detect the infection at the early stage of infection or by Lyssavirus detected in saliva samples from livestock that may be infected and are just days away from death It has the advantage of being able to diagnose infectious diseases. Real 11i:, Na primer - and Nb primers as defined in the present invention (this is located within the N gene and It is not very far away (400 dp), and there are very few 'J saviruses. can be detected even when the sample is not present and even if the sample is highly degraded. (Yes/No response).

In fact, the sensitivity of conventional methods is lower than that of the method according to the invention; is rapid and can be diagnosed in less than 12 hours.

(Below margin continues on next page) Example 10 Expression of Mokola virus G protein Cloning of Mokola genome The insert fragments pMD10 and pMA10 induced by It spans 1/2 of the protein gene. These inserts have in common Bgl I [restriction site spacing 6830 genomic 3' nucleotides can bind to the single insert fragment pM7 containing From now on, G sugar protein The cDNA of the protein gene is created by combining the restriction enzymes FnuD If and SspI. The polyhetrin gene of baculovirus can be isolated by double digestion. The inside of the 5゛ promoter region and especially that 3 corresponding to the polyadenylation signal. ’ area. This construct is placed inside the pUC vector to allow for propagation. Prepared in 1 part. Next, it is cultured in suspension with wild-type baculovirus. Caterpillar Subodobutella frugiperda S f 9 (spodop terafrugiperda) cells. G sugar tongue By double recombination, the protein gene is inserted in its place in the wild-type viral polyhetrin. inserted into. After cloning, the recombinant baculovirus plaques are Wild-type virus plaques, found in minerals with a vague appearance, can be easily distinguished from its refractive appearance.

Several selected recombinant clones were used to infect caterpillar cell cultures. there was. Three days after infection, total protein cell extracts were prepared. Mokola sugar An additional protein band similar in size to that of the protein may be a recombinant clone. was observed only in extracts derived from For further testing of this additional protein, For this purpose, other cultures were removed from methionine and supplemented with labeled methionine. Infected in culture. After that, G-glycoprotein appeared more intensely, and indeed, When labeled, the culture ↑ shows polybebutydia that were expressed to a high degree (Figure 5 a).

Figure 5b shows the negative control.

Mokola virus glycoproteins on the surface of cells infected with recombinant baculovirus. An illustration of the expression of a recombinant baculovirus expressing the Mokola virus glycoprotein. demonstrated on Subodobutella frugiperda cells after infection with S. frugiperda. that cell They were fixed in acetone for 30 minutes at -20''C. Incubated with monoclonal antibody against glycoprotein (Acm) .

After washing, the generation is coupled to fluorescein isothiocyanate (ITCF). This was done by incubating with an anti-immunoglobulin antibody from a mouse that had been tested.

After washing, images were observed using a microscope under ultraviolet excitation (Figures 5a and 5b).

The above glycoprotein also has a fluorescent moiety specific for Mokola virus glycoprotein. Noclonal antibodies allow recombinant virus to be clearly detected on the surface of infected cells. In Figure 6, Lane! , 2 and 3 are mokola virus G-tang Subodoptera after infection with a recombinant baculovirus expressing the protein. - Compatible with Frugiperda. Also, the arrow indicates the position of the 6w protein gel. and lane T corresponds to the negative control.

Example 11 Demonstration of immunity of Mokola virus glycoprotein (G, MOK) Subodoptera frugiberda (SF9), II, which is susceptible to baculovirus, G, baculovirus expressing MOK or polyhetrin gene (NPEV The cells were infected with a wild-type baculovirus that does not express C.clel. 3 after being infected After a day, the cells were washed with PBS buffer. Then, a variety of these cells Test the protective value of this cell suspension in mice by dilution and modified barbell test method. I went to do it. Groups of 10 mice were vaccinated intraperitoneally twice, one week apart. Two weeks after the first vaccination, various virus dilutions were administered into the brain. inoculated and infected. And it's been 4 weeks since the brain infection of these mice! After I observed. After this period ended, specific mortality was calculated.

Figure 9 shows that non-vaccinated [(NV) mice and NPEVdel-infected SF9 cells were vaccinated. This shows that the mortality curves of mice inoculated with Chin are hardly different. In other words, NPEVdel-infected SF9 cells show no non-specific protection in mice.

Figure 10 shows that the vaccine dose of 100,000 5FE9-G, MOK cells was 10'5FE9-G, MOK cells are more than 2.5 This shows that it is sufficient to obtain a high defense i11 index.

As is clear from the foregoing, the present invention is more clearly Without being limited in any way to the embodiment or application, it may be necessary to Specialization in this field (below margin continues on next page)

Claims (1)

[Claims] 1. Extract the viral RNA and/or transcripts of any lyssaviruses present. (1) To obtain cDNA from genomic RNA or mRNA, use product-specific primers, in the case of lyssavirus serotypes, lyssavirus-specific primers and all Conservation of genes common to viruses (2) then contacting the cDNA sequence with at least one primer suitable for Lyssavirus selected from the primers consisting of the entire sequence (hereinafter referred to as multivalent primers), in order to amplify at least one fragment of the cDNA; a pair of suitable primers for Lysvirus (provided that one of the primers is different from that in step (1) and the other primer is the same or different from that in step (1)); (3) then the amplified cDNA sequence; detection by appropriate means, rapid detection of small amounts of lyssaviruses present in characterized biological samples and/or is a method of identification. 2. One of the primers was used to detect rabies and 3' rabies localized at positions 1-18 of the Mokola genome. The method according to claim 1, which is a canine disease primer. 3. One of the primers is M2 rabies localized at positions 2901-2918 of the rabies genome. The method according to claim 1, which is a canine disease primer. 4. One of the primers was used to target positions 4665-4687 of the rabies genome and 2. The method according to claim 1, wherein the rabies primer consists of 23 nucleotides localized at positions 4675-4697 of the genome, hereinafter referred to as G primer. 5. 2. The method according to claim 1, wherein one of the primers is a rabies primer consisting of 24 nucleotides localized at positions 5520-5543 of the rabies genome and positions 5545-5568 of the Mokola genome, hereinafter referred to as L primer. 6. One of the primers is the Mokola primer, which consists of 18 nucleotides localized at positions 587-605 of the rabies and Mokola genome, and is hereinafter referred to as the Na primer. A method according to claim 1. 7. 2. The method according to claim 1, wherein one of the primers is the rabies and Mokola primer consisting of 16 nucleotides localized at positions 1013-1029 of the Mokola genome, hereinafter referred to as Nb primer. 8. Detection of amplified nucleotide sequences is a suitable probe for various lyssaviruses. What is claimed in claims 1 to 7 that is carried out by hybridization by means of a probe or a series of probes? One of these methods. 9. Detection of the amplified nucleotide sequence involves digesting the sequence with at least one suitable restriction enzyme and then separating the resulting fragments by electrophoresis. 8. A method according to any one of claims 1 to 7, which is carried out by. 10. The series of enzymes are BanHl, HindII, HindIII and Pstf. 10. The method according to claim 9, comprising: 11. A series of enzymes consists of Rsal, TagLBstxl and Fnu4Hl. Method according to requirement 9. 12. cDNA nucleotide sequence from the genomic RNA of Mokoralisavirus, which consists of approximately 12,000 nucleotides and whose non-coding sequence is: vinegar. 13. The 3' and 5' ends are complementary, and the 12 nucleotides at the 5' end are the rabies virus. identical to that of the standard PV species, with a “leader” sequence from 3′ to 5′ The gene encoding SRNA and then the proteins N, M1, M2, G and L. 13. The nucleotide sequence according to claim 12, comprising a gene comprising: 14. The following nucleotide sequence and derived amino acid sequence (Ia-Ig), - from nucleotide 1 to nucleotide 5528: Leader sequence RNA [Sequence is available] [Sequence is available] - From nucleotide 5588 to nucleotide 5633 [Sequence is available] - From nucleotide 5635 The Mokola virus according to claim 12 or 13, consisting of nucleotide 6017 [sequence available] - nucleotide 6020 to nucleotide 6831 [sequence provided] - nucleotide 8641 to nucleotide 8703 [sequence provided] - nucleotide 11525 to nucleotide 11939 [sequence provided] cDNA sequence from genomic RNA. 15. 15. A fragment of a sequence according to any one of claims 12 to 14 encoding N nucleoprotein and corresponding to nucleotides 71-1420 of the cDNA sequence. 16. 15. A fragment of the sequence according to any one of claims 12 to 14 encoding the M1 protein and corresponding to nucleotides 1524 to 2432 of the cDNA sequence. 17. 15. A fragment of the sequence according to any one of claims 12 to 14 encoding the M2 protein and corresponding to nucleotides 2516 to 3121 of the cDNA sequence. 18. 15. A fragment according to any one of claims 12 to 14 encoding a G protein and corresponding to nucleotides 3328-4893 of the cDNA sequence. 19. A fragment according to any one of claims 12 to K encoding the NH2 terminus of the L protein and corresponding to nucleotides 54 43-528, 5588-5633, 5635-6017 and 6021-6831 of the cDNA sequence. 20. 15. A fragment according to any one of claims 12 to 14, being a non-coding fragment of said sequence and corresponding to 4897-5442 of said sequence. 21. It is a non-segmented, non-polyadenylation-negative, single-stranded RNA of approximately 12,000 nucleotides with a 3' to 5' "leader" sequence. Genes encoding SRNA and then N nuclear protein, M1 phosphoprotein, M2 matrix [There is a sequence] A non-coding gene called the ps1 coagulase gene and a gene encoding the L polymerase protein. The Mokola virus genome RNA sequence contains a gene whose genome is always bound to the N nucleoprotein. 22. From the 3' end, five consecutive monocistronic fragments encoding proteins N, M1, M2, G and L, namely - the fragment corresponding to nucleotides 59-1484 of the sequence of formula I in conjunction with polyA; - a fragment corresponding to nucleotides 1495-2489 of the sequence of formula I combined with polyA; - a fragment corresponding to nucleotides 2501-3283 of the sequence of formula I combined with polyA; corresponding fragments, - fragments corresponding to nucleotides 330 7-5380 of the sequence of formula I, which binds polyA and codes for the G protein, - sequences 5416-5633, 56 35-6017, 6020-6831, 8641, 8703, 8809-9217, and a fragment encoding the L protein consisting of 11525-11858, - and bicistronic M1-M2. thing. 23. The entire genome from the 3' end to the 5' end, i.e. -4150 nucleotides and contains the "leader" sequence RNA, N nucleoprotein, M1 protein, M2 protein. -2850 nucleotides and corresponds to a fragment of a sequence encoding a G protein and a fragment of a sequence encoding a G protein. The fragment, which corresponds to the fragment encoding the L gene and the L gene, consists of 6830 nucleotides from the −3′ end and encodes the leader sequence RNA, proteins N, M1, M2 and G, and the first 1420 nucleotides of the L gene ( (hereinafter referred to as pM7) into the Bg1II site. A fragment obtained by ligation of input pMD10 and pMA10, - consisting of about 3300 nucleotides, designated pMB5, and corresponding to a fragment of the sequence encoding L protein, - consisting of about 2800 nucleotides (hereinafter referred to as pM12). , a fragment corresponding to the fragment encoding L protein. - Consists of approximately 700 nucleotides (hereinafter referred to as pMR15a) and encodes the L protein. fragment of the sequence to be encoded as well as the fragment corresponding to the non-transcribed 5' end of the genome. fragment, which fragments, when separated, specifically hybridize to RNA fragments or cDNA fragments derived from transcription or replication of the Mokola genome, respectively. A cDNA clone from Mokola virus genomic RNA that exhibits the ability to hybridize. loan. 24. Collection National de Cultue at Institut Bastour 24. The cDNA clone designated pM7 according to claim 23, deposited with Ledu Microorganisme (CNCM) on March 22, 1989 as No. 1-847. 25. 24. The cDNA clone named pMB et al. according to claim 23, which has been deposited with said depository institution on March 22, 1989, number 1-848. 26. Deposited with the said depositary institution on March 22, 1989 as No. 1-849. 24. A cDNA clone according to claim 23. 27. Deposited with the said depositary institution on March 22, 1989 as No. 1-850. 24. A cDNA clone according to claim 23. 28. A nucleotide probe consisting of a nucleotide sequence according to any one of claims 12 to 27 or a fragment thereof labeled with a marker such as a radioisotope, a suitable enzyme or a fluorochrome. 29. Nucleotide according to claim 23 corresponding to site 4675-5568 or a fragment thereof, in particular fragment 4897-5442 or its complementary strand. Doprobe. 30. Nucleotide sequence, flag according to any one of claims 12 to 27 A peptide or compound encoded by a fragment or a combination of several fragments. Fragment of putide. 31. It is encoded by the N nucleoprotein gene and has the amino acid sequence of formula II below. 31. The peptide according to claim 30, having the sequence: 32. It is encoded by the M1 protein gene and has the amino acid sequence of formula III below. 31. A peptide according to claim 30 having the sequence: 33. 31. The peptide according to claim 30, which is encoded by the M2 protein gene and has the amino acid composition of formula IV below. 34. 31. The peptide according to claim 30, encoded by the G-glycoprotein gene and characterized by the following formula Va and Vb amino acid sequence: (Va) (Vb): 35. The sequence of formula VIa encodes the L protein gene and has one of the following amino acid sequences: Sequence of formula VIb: Sequence of formula VIc: Sequence of formula VId Sequence: [Sequence is present] Peptide according to claim 30. 36. Peptide according to any one of claims 30 to 35, which is obtained by synthesis. peptides and/or fragments thereof. 37. at least one nucleotide sequence according to any one of claims 12 to 27. A vector containing a sequence or part of a sequence thereof. 38. Even without creating a natural product of baculovirus (1) Expression preparation of baculovirus section sequence and (2) the remains of Mokola virus according to any one of claims 13 to 28. a polylinker suitable for inserting a gene or at least a fragment of a gene; mokorovirus obtained by homologous recombination of an appropriate shuttle vector consisting of Expression vectors for peptides, fragments or combinations of fragments of -. 39. Is the shuttle vector (1) the 5' control region of the polyhedrin gene? (2) a mocorovirus gene or at least a fragment thereof; (3) a 3' control sequence, in particular containing a polyhedrin polyadenylation site, the vector being capable of propagating the construct; 39. An expression vector according to claim 38. 40. 39. The G glycoprotein gene of Mokola virus or a fragment thereof is inserted at the level of a polylinker under the control of a polyhedrin gene promoter so as to obtain an expression vector charged with said glycoprotein. Expression vector by. 41. Deposited with the said depositary institution on March 22, 1989 as No. I-851. 41. An expression vector according to claim 40. 42. The N nucleoprotein gene or its fragment is linked to the polylinker at the level of the polylinker. The expression vector according to claim 38 or 39, wherein the expression vector is inserted under the control of the hedrin gene promoter so as to obtain an expression vector charged with the protein. 43. at least one peptide and/or according to any one of claims 30 to 36 is a combination of the fragment and optionally at least one pharmaceutically acceptable vehicle. A vaccine against mocorovirus for humans and/or livestock. 44. 44. A vaccine according to claim 43, consisting of a G-glycoprotein and/or a fragment thereof, and/or an N-nucleoprotein and/or a fragment thereof. 45. Lysvirus multivalent vaccine consisting of at least one peptide and/or fragment thereof according to any one of claims 30 to 36 and optionally at least one peptide and/or fragment thereof of at least one other Lysvirus serotype. . 46. Peptides and/or fragments thereof are present in suitable excipients and/or acceptable Advantageously combined with adjuvants, especially conventional adjuvants for human and livestock vaccines. 46. A vaccine according to any one of claims 43 to 45. 47. obtained by immunizing a mammal, especially a rodent, or even a mouse, with the peptide and/or fragment thereof according to any one of claims 30 to 36. Monocloners specific for Mokola virus peptides and/or fragments thereof antibody. 48. The anti-Mokola antibodies that may be present in the biological sample are contacted with the peptide or fragment thereof according to any one of claims 30 to 36, the anti-Mokola antibodies are allowed to bind, if present in the biological sample, and the result is analyzed, particularly EIA, RIA and fluorescence An immunization method for detecting antibodies to anti-Mokola virus peptides and fragments thereof, which comprises detecting the antibodies by an appropriate means such as: 49. 49. A method according to claim 48, wherein said peptide or fragment thereof is immobilized on a suitable solid carrier. 50. When the sandwich method is used, the appropriately labeled antibody to be assayed is 50. The method according to claim 48 or 49, wherein the method is carried out using a second antibody directed against. 51. Claim 28 or claim 28, wherein Mokola virus that may be present in the biological sample is appropriately treated. is contacted with at least one nucleotide probe according to 29, in which Mokola virus genomic RNA and/or transcripts are present in the sample. A quick and special test for the Mokola virus, which consists of combining the How to detect abnormalities. 52. Useful amounts of buffers and reagents appropriate to carry out the detection, and appropriate amounts of at least two appropriate primers, an appropriate amount of at least one nucleotide probe and an appropriate amount of at least one nucleotide probe. A simple kit for carrying out the method for detecting and/or immobilizing at least one lyssavirus according to any one of claims 1 to 11, further comprising one restriction enzyme. 53. at least one peptide and/or according to any one of claims 31 to 37 the appropriate amount of that fragment, and the least useful amount of a suitable buffer for which detection is to be carried out. The antimony in a biological sample according to any one of claims 43 to 50, comprising: A simple kit for performing the Kola antibody measurement method. 54. 54. A kit according to claim 53, wherein the peptide is a G-glycoprotein according to claim 35 or 37, or a fragment thereof, immobilized on a suitable immobilization carrier. 55. 54. A kit according to claim 53, wherein the peptide is an N-nucleoprotein or a fragment thereof according to claim 31 or 36, immobilized on a suitable solid carrier. 56. At least one probe and or nucleotide according to claim 29 or 30 53. A simple kit for carrying out the method for detecting Mokola virus according to claim 52, which comprises at least an appropriate amount of a fragment of a deprobe and a useful amount of a buffer suitable for carrying out the detection.