The nucleotide sequence of the virus of classical swine fever (csfv) (options), polypeptide csfv, pestivirus vaccine against csfv, diagnostic kit and method

 

(57) Abstract:

The invention relates to biotechnology, Virology and immunology, and can be used to create a vaccine against the virus of classical swine fever (CSFV). The nucleotide sequence corresponding to the genome of the virus of classical swine fever or mutant represents the nucleotide sequence of the C-strain virus classical swine fever specified in sequence ID 1, the complement, or RNA equivalent of such a nucleotide sequence, or contains the nucleotide sequence encoding the amino acid sequence 268-494 in the sequence with ID 1 and/or sequence that carries a mutation in the nucleotide sequence that encodes the amino acid 690-877, or the nucleotide sequence encoding amino acids 690-1063 sequence 1, and optionally containing a mutation in the amino acid sequence 268-494. Mutation selected from deletions or substitutions of one or more amino acids, altering at least one epitope. The resulting polynucleotide part of a diagnostic kit and pestiviruses vaccine virus classicianus classical swine fever. 5 S. and 9 C.p. f-crystals, 5 Il., 8 table.

The technical field to which the invention relates

The object of the present invention is a method of creating a full-size DNA copy of the genome of the C-strain vaccine strain of classical swine fever and transcription of its RNA, which after transfection into cells induces the synthesis of infectious virus-strain. This invention also includes (protivostoyanie) vaccine derived from the C-strain, as well as subunit vaccines pestivirus, tools, and methods of diagnosis pestivirus infections. The present invention further includes a method of detecting immunoactive substances in the sample by using competitive analysis.

Prior art

Classical swine fever is a highly contagious disease that often causes the death of pigs from a severe fever, and bleeding, and may occur in acute or chronic form (Van Dirschot, 1986, Hog cholera, pages 289-300. In the book Diseases of Swine", Jowa State University Press, Ames ). Outbreaks occur periodically in different countries of Europe and other regions and can cause large economic losses.

Vaccination of piglets vaccine strain of live attenuated virus classic with the th fever (Terpstra and Wensvoort, 1988, Vet. Environ, 16: 123-128). The main disadvantage of vaccination of pigs with routine vaccines, one of which is the C-strain is the fact that vaccinated pigs impossible to distinguish by serologic methods from pigs infected with the wild strain of the virus of classical swine fever. However, the strain is considered to be one of the most effective and safe live vaccines. Inclusion (serum) token in the C-strain provides significant benefits greatly improves this vaccine.

The virus of classical swine fever (SFV belongs to the genus Pestivirus species Flaviviridae (Francki, R. I. B and others, 1991, Flaviviridae, pages 223-233 in the fifth report of the International Committee on taxonomy of viruses, Archiv. Virol. Suppl. 2, Springer Verlag, Vienna). Two other members of the genus Pestivirus, in which structural, antigenic and genetic closely associated with the virus of classical swine fever are viral diarrhea virus in cattle (BVDV ) and virus diseases of the Border (BDV), which affects mainly sheep (Moennig and Plagemann, 1992, Adv., Uirus Res., 41:53-98; Magtape and others , 1990, Virology 177: 184-198; Becher and others, 1994, Virology 198: 542-551).

The genomes of pestiviruses contain an RNA molecule with a positive chain length of 12.5 thousand pairs of nucleotides (Renard and others, 1985, DN 4:429-438; Moormann and Hulst 1988, Virus. Res. , 11 is the viral diarrhea virus in cattle can be much larger ( Meyers and others, 1991, Virology, 180: 602-616; rs and others, 1992, Virology, 191: 368-386; Qi and others, 1992, Virology, 189: 285-292).

A distinctive feature of viruses with RNA genome with positive chain is that their genomic RNA is infectious, i.e. after transfection of this RNA in cells, providing a replication of the virus, to form infectious virus. As expected, genomic (viral) RNA pestiviruses is also infectious (Moenning and Plagemann) 1992, Adv. Virus Res., 41: 53-98).

Currently, recombinant DNA technology makes it possible for in vitro transcription of cloned DNA. This allowed us to synthesize infectious RNA in vitro from a DNA copy of the genome of RNA virus with a positive circuit. In the field of molecular engineering is well known that DNA in contrast to RNA is amenable to site-directed mutagenesis. Therefore, the technology of synthetic infectious RNA significantly expanded research in the field of replication, virulence, pathogenesis, recombination, RNA, forming vectors and anti-virus strategies of RNA viruses with a positive circuit. However, application of this technology may cause serious problems. They were described in a recently published review Boyer, Nauni, 1994 ( Karpova et al., 198: 415-426). Daopei genome of the virus, containing RNA with a positive circuit, and upon receipt of synthetic infectious RNA from a full-size DNA copy.

A brief statement of the substance of the invention

The present invention provides a nucleotide sequence corresponding to the genome of the virus of classical swine fever, which contain at least part of the nucleotide sequence of the C-strain virus, classical swine fever, presented in the form of sequences with identity 1, the complement or equivalent RNA such nucleotide sequences or their mutants. Also provided degenerative nucleotide sequences that have different nucleotides, but encode the same amino acids. In the scope of the present invention also includes polypeptides encoded by these nucleotide sequences, and vaccine strains whose genome contains a nucleotide sequence, in particular a recombinant strain of the virus on the basis of transcripts of full-length DNA copy of the genome of strain of the virus of classical swine fever.

As mentioned above, are also useful partial nucleotide sequence in casavalle, the coding amino acids 1-1063 sequence with ID 1. The mutation may be a substitution of the relevant part of the genome of another pestiviruses strain, the substitution of one or more amino acids or deletions. The mutation can also be inserted or replaced by a heterologous nucleotide sequence that changes the strategy of translation of the nucleotide sequence of the virus of classical swine fever or processing of the polypeptide encoded by the nucleotide sequence of the virus of classical swine fever. In addition, the mutation may be an inserted or replaced by a heterologous nucleotide sequence encoding a polypeptide that induces immunity against another pathogen; in this case, the sequence of the virus of classical swine fever is used as a vector for heteroanalogues.

The present invention includes the nucleotide sequence pestiviruses genome as a whole and parts thereof or a mutant sequence containing a mutation in Subplate protein E1, that is, in the nucleotide sequence encoding amino acids corresponding to and the and nucleotide sequences. These polypeptides are particularly useful for the protection of animals from pestiviruses infection due to the fact that they can be used for diagnostic purposes for differences between animals infected with wild strains of pestivirus from vaccinated animals.

In addition, the scope of this invention includes a vaccine containing the nucleotide sequence, polypeptide or vaccinal strain, and diagnostic compositions containing the nucleotide sequence or the polypeptide described above, or an antibody induced against such a polypeptide.

This izobreteniya also includes the methods and means of diagnostics pestivirus infections, such means and methods, which allow to differentiate infected animals from vaccinated.

This invention also provides a method of determining test agent such as an antibody or antigen in the immunoassay, which is developed on the basis of a test of specific binding using immobilized reference substances for specific binding and similar labeled reference substance.

A detailed description of the invention
<(C-strain; the application for the European patent 351901) virus classical swine fever. This allows you to create full-size DNA copy of this sequence, on the basis of which you can transcribing synthetic RNA, which, after transfection of the respective cells, in particular cells S6-M ( Kas Z. and others, 1972, Res., Vet., Sci., 13: 46-51; the application for the European patent 351901), causes the synthesis of infectious virus-strain. Below is a description of this discovery with the aim of obtaining vaccines based on modified C-strain, such as vaccines containing (serological) marker. Although this invention is illustrated in relation to one strain of the virus of classical swine fever, it is also useful and applicable for pestivirus strains as a result of the exchange of specific genomic segments, described below, between other pestiviruses and C-strain virus classical swine fever or through the creation of "infectious" DNA copy another pestivirus.

The nucleotide sequence of a DNA copy of the genomic RNA of the C-strain is represented as a sequence with ID 1. All figures in the text refer to this sequence and may vary slightly from the other sequences is cityfone, consisting of 11694 nucleotides encoding polyprotein from 3898 amino acids. The size of the open reading frame similar to the size of the genomes of strains Brescia ( Moormann and others, 1990, Virology, 177: 184-198) and Fi (Meyers and others, 1989, Virology, 171: 555-567) virus classical swine fever.

The open reading frame begins with an ATG at positions of nucleotides 374-376 and ends with a TGA codon in the positions of nucleotides 12068-12070. The length of the 5'end non-coding region preceding an open reading frame, is 373 nucleotides. This sequence is a highly conserved region in strains of Brescia, Alfort and C (Fig. 2) and the predicted secondary structure of this region are similar to the structure of the 5'-terminal noncoding region of hepatitis C virus ( Brown and others, 1992, Nucleic Acids Res., 20: 5041-5045), which is another member of the family Flaviviridae. It was found that the 5'-end noncoding region of hepatitis C virus contains aminoallyl website internal ribosome ( Tsukiyama-Kohara and others, 1992, J. Virol., 66:1467-1483). These sites have important regulatory functions (Agl. , 1991. Adv. Virus Res., 40: 103-180). The analogy with the hepatitis C virus shows that the 5'-end non-coding region of the virus of classical swine fever also contains aminoallyl website predstavljaet an important regulatory element. Aminoallyl website internal ribosome can be used as a site for mutations with the aim of weakening the virus, but also changes the translation of the open reading frame.

The second important region that regulates replication pestiviruses, is a 3'-terminal non-coding region. A comparative sequence analysis of strains with sequences of strains Bresci and access in this area was discovered a sequence of 13 nucleotides, which is characteristic for C-strain (Fig. 2B). This unique sequence TTTTTTTTTTTTT occupies the position of the nucleotides 12128-12140 in the sequence with ID 1. This is the only box of more than two nucleotides in a row, found in the sequence of the C-strain, compared with the sequences of strains Brescia and access. In the rest of the sequence in the 3'-terminal non-coding regions of the three strains of the virus of classical swine fever are sufficiently homologous. The overall homology of sequences in this region is lower when comparing strains of the virus of classical swine fever virus and viral diarrhea in cattle. However, it is obvious that the sequence TTT viral diarrhea in cattle. Therefore, the sequence TTTTCTTTTTTTT is characteristic of the genome of strain and can serve as an excellent marker for a specific sequence of strain. This sequence can be used as the basis for nucleotide probes and to determine the sequences to identify specific pestiviruses C-strain. Therefore, we can conclude that all pestivirus strains that have this sequence in the 3'end non-coding region (not necessarily in the same position as in the C-strain), refer to the C-strain and are included in the scope of the present invention.

An important parameter of the infectivity of the transcripts of a DNA copy of the genome of pestivirus is the amino acid sequence. In this connection it is necessary to consider two aspects of cloning and sequencing RNA-containing viruses in General and pestiviruses in particular. First, the frequency of mutations of the genome of viruses containing RNA with a positive circuit is quite high (about 1104nucleotides during replication), so none of the viral preparations or preparations of viral RNA is not clonal in relation to viral RNA, which it contains. Among these RNA molecules Etop-codons in a large open reading frame, it's easy to recognize. In addition, it is possible to detect mutations in the active sites of viral enzymes or known structures of proteins. However, if the relationship between amino acid sequence, the function or structure of the protein is unknown, which is typical for most pestivirus proteins, it is impossible to predict which amino acid is correct, and what isn't. Secondly, mutations can occur during the synthesis of cDNA. Therefore, the genome of strain cloned and sequenced twice independently from each other. Areas with dissimilar sequences were cloned and sequenced at least three times (cf. Fig. 1). The sequence, which had met twice in a certain position, was considered correct. The necessity of this approach for the synthesis of infectious transcripts of a DNA copy of the genome of strain is confirmed by the next discovery.Full-size DNA copy pPRKr1c-113, obtained after the second series of cloning and sequencing (Fig. 3) turned out to be non-infectious. After cloning and sequencing of regions with dissimilar sequences of cDNA clones obtained during the first and second series, we found five amino acids that differed in the full-size copies Verlot in pPRKf1c-113 was obtained clone pPRKf1c-133, which synthesized infectious transcripts (Fig. 4). Five differences are in positions of amino acids 1411 (Vl --> A1A); 2718 (G1 --> sp); 2877 (Val --> Met ); 3228 (Leu --> Met ); 3278 (Tight --> Glu). Amino acids encoded in these provisions of the cDNA sequence, which is non-infectious, indicated to the arrow, and amino acids that are in the same positions infectious copies of the following arrow (sequence ID 1). Will become.if the change of each amino acid separately by the lack of infectivity of DNA-copies-strain must be determined by analysis of the infectivity of transcripts of individual mutations in each of these five amino acids. However, the result shows that even small differences in amino acid sequence can be important for infectivity of transcripts of a DNA copy of the genome of strain. He also suggests that the creation of infectious transcripts copies of the sequence pestivirus may be in practice impossible because of the small differences in the sequences (even at the level of single amino acids), which may be unsubstituted.

Mutants derived from the C-strain, which is IGNOU sequences with identity 1 such mutations, as deletions, insertions, (several) mutation of a nucleotide and / or inserted and/or replaced genomic fragments taken from other pestivirus strains.

The sequence of strains can be divided into four areas suitable for mutation and/or exchange. The first region is the 5'-terminal non-coding sequence, okutyvaya nucleotides 1 to 373. The second region encodes structural proteins Npro-C-E2-E3-E1 and includes nucleotides with 374 on 3563. The third region encodes non-structural proteins and encompasses nucleotides with 3564 on 12068. The fourth area is a 3'-terminal non-coding sequence that comprises nucleotides with 12069 on 12311.

One area that is particularly suitable for the production of vaccines-markers-strain represents a genomic region encoding the structural proteins Npro-S-E2-E3-E1. This region is located between amino acids 1 and 1063 in the sequence with ID 1. Preferred Subplate this part of the genome defined by the following amino acid sequences 1-168 (Npro), 169-267 (C), 286-494 (E2), 495-689 (E3) and 690-1063 (E1) or their parts. For example, N-terminal antigenic part of the region that encodes a protein E1-piece is Synthesized derivative of strain is contagious and it can be distinguished from wild-type strain and the strain Brsi by reaction with specific monoclonal antibodies-strain and strain Brsi affecting proteins E1 and E2; for example, the s-strain was subjected to interaction with monoclonal antibodies specific for the protein E1 strain Brsi (table 1). The antigenic properties of the new mutant has changed compared with the parent virus, and this suggests that replacement of the N-terminal half of the protein E1 C-strain of the same area of another strain of the virus classical swine fever is one of the possible ways of creating a vaccine-token-strain. However, this invention is not limited to replacement of the N-terminal halves of the protein E1 in the C-strain and other strains of the virus of classical swine fever. N-terminal half of the protein E1 from any other pestiviruses strain can be replaced by the corresponding parts of the E1 protein of the C-strain. In this respect, particularly useful are the protein sequence E1 pestivirus strains that were isolated from pigs, but refer to antigenic group, which is not included With the strain. Examples of such strains, which are selected on the basis of cross-neutralization, are the strains of "HER van," Stam ", "SFUK 87"of pigs in the European Council. 16-17 June 1992 V1/4059/92-EN(PV ET/EN/1479), 1992, pp. 59-62).

It was found that N-terminal half of the protein E1 contains three distinct antigenic domain a, b and C, which are located in different parts of the E1 protein, and each of them interacts with a strongly neutralizing monoclonal antibodies (Wensvoort, 1989, J. Gen. Virol., 70: 2865-2876; Van Rign and others, 1992, Vet. Environ., 33: 221-230; Van Rign and others, 1993. J. Gen. Virol. , 74: 2053-2060). Epitopes that are stored in 94 the tested strains of the virus of classical swine fever, mapped in domain a, while the epitopes of domains b and C are not saved (Wensvoort 1989, J. Gen. Virol. , 70: 2865-2876). Mapping of epitopes hybrids E1 genes of strains of Brescia and C (Van Rign and others, 1992, Vet. Environ., 33:221-230) and deletion mutants of the protein E1 strain Brescia, suggests that the domains a and b + C form two different antigenic complex in the N-terminal half of the protein El (Van Rijn and others, 1993, J. Gen. Virol., 74: 2053-2060). This assumption was further confirmed by the discovery that six cysteines under the provisions 693, 737, 792, 818, 828 856 N-terminal half of E1 are important for correct protein folding E1. However, at least Cys 792 no significant effect on the infectivity of strain Brescia, as a mutant of the virus that are resistant to the hcpa is the arrival at the 9th International Congress on Virology 8-13 August, Glasgow, Scotland).

If small changes in amino acid sequence can stand the reason for the loss of infectivity of the RNA-strain (see example 2), the replacement of cysteine in position 792 indicates that replacement of the amino acid in position, which is much less suitable for modification without loss of function may result in a viable virus mutant. Thus, the effect of replacement of certain amino acids on the properties of the virus, it is necessary to determine empirically for each amino acid in the sequence of strain C. This shows again that on the basis of previously published data, it is impossible to determine a target sequence for modification of the C-strain, for example, to create a vaccine token.

Important to create vaccines-markers-strain has the ability to differentiate serological methods vaccinated pigs and pigs infected with wild virus strain of classical swine fever. Previously it was shown that the vector of the live attenuated virus pseudoleskeella expressing the E1 protein, or immunoaffinity purified E1 protein expressed in insect cells using the baculovirus vecto the J. Virol., 67:5435-5442). It was found that the mutant protein E1 with the remote domain And remote domain + (Fig. 5) also induce a protective immune response in pigs against cholera (table 2). This suggests that protective immunity induced by a vaccine strain does not depend on neutralizing antibodies against domains a and b + C. Therefore, the mutants pestiviruses strain, have replaced or mutated only domain a or domain b + C or part thereof with the corresponding region of another pestivirus, preferably, but not necessarily of pestiviruses isolated from pigs, which is one and the other antigenic group compared with the C-strain (examples above), also included in the scope of the present invention. Region E1 protein containing the domain and suitable for exchange or mutation, is located between amino acids and 870 785. In addition, can be replaced or motivovany part of this area, such as Subplate located between amino acids 785 and 830, as well as between amino acids and 870 829. Region E1 protein comprising domains b + C and a suitable replacement or mutation, located between amino acid 691 and 750. Can also be replaced or motivovany part of this area, such as Subplate, located the moustache, antibodies against the E2 protein (Kwang and others, 1992, Vet. Environ., 32: 281-292; Wensvoort, unpublished observations). Therefore, the second region, suitable for a vaccine (token) by mutations (deletions, insertions, point mutations) or replacing the relevant genetic material pestiviruses with other antigens or pestiviruses related to other antigenic group, is a region encoding a protein E2.

With a strain can also be used as a vector for insertion or expression of heterologous genetic material (sequences). In the case of vector synthesis of heterologous genetic material introduced in the C-strain is used to change strategy broadcast a large open reading frame and processing polyprotein, encoded by this open reading frame. An example of a sequence that is suitable for policy changes, broadcast a large open reading frame is the sequence that defines aminoallyl website internal ribosome (Duke and others, 1992, J. Virol. , 66: 1602-1609, and here opposed to materials). An example of a sequence that is suitable for modifying the processing of polyprotein, the signal is consistently domesticsale reticulum ( Blobel, 1980, Proc. Natl. Acad. Sci., USA. 77: 1496-1500; Kreil, 1981, Annu. Rev. Biochem., 50: 317 - 348). The signal sequence is broken down by cellular signal peptidases. However, the sequence encoding the cleavage sites of viral proteases, can also be used to modify the processing of polyprotein.

Sequences that have been introduced and expressed by the vector-strain, can be used as a marker for identification of vaccinated pigs or to protect them against the pathogen from which was taken heterologous insertiona sequence. Sequence markers are preferably antigenic and refer to microorganisms that are not can replicate in the body of pigs. They can encode the full gene products (e.g., capsid proteins or membrane) or antigenic parts (e.g., epitopes). Sequence markers are preferably derived from a virus belonging to the following families: Adenoviridae, Arenaviridae, Arteriviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Rhabdoviridae, Parvoviridae, from the poxviridae, Picornaviridae, Reoviridae, Retroviridae and Togaviddae.

However, sequence markers can also encode artificial antigens, not sophila, alkaline phosphatases of human placenta, the luciferase fireflies and chloramphenicol-acetyltransferase.

Heterologous genetic material encoding one or more proteins, creating a protective immunity against a disease caused by a pathogen, appropriate heterologous genetic material can be obtained from other pestivirus strains, including sequences of the above strains, parvovirus, swine respiratory coronavirus pigs, virus, infectious gastroenteritis, respiratory syndrome virus of swine (Lelystad virus, the European patent 92200781.0), virus Aujeszky's disease virus (false rabies), virus endemic diarrhea in pigs and such bacteria as Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Streptococcus suis, Treponema hyodysenteria, Escherichia coli, Leptospira, and microplasmin, such as M. hyopneumoniae and M. lyorhinis.

Sites suitable for insertion of heterologous sequences in the C-strain, but not the only possible one, located between amino acid residues 170 and 171, between residues 690 and 691 and between residues 691 and 692 and is listed in the sequence with ID 1.

The present invention also includes diagnostic tests, to, aderasa (mutated) protein E1 and/or (mutated) protein E2, from pigs infected with the wild strain pestivirus. Acceptable forms of such diagnostic tests are described in examples 4 and 5. Conventional non-selective enzyme-linked immunosorbent assay (ELIS) virus classical swine fever were performed using E1 protein as antigen when performing complex trapping-lock in accordance with the description given by Sensorcom and others, 1988 (Vet. Environ., 17: 129-140). In the prototype enzyme-linked immunosorbent analysis of complex trapping-blocking (CTB-ELISA), also known as enzyme-linked immunosorbent assay with the lock in the liquid phase or solid-phase enzyme-linked immunosorbent assay with a two-layer immunoanalyzer, using two monoclonal antibodies generated against the protein E1 strain Brescia virus classical swine fever. The epitope for monoclonal antibody b3, which is located in domain a, And is stored in the strains of the virus of classical swine fever, while the epitope of the monoclonal antibody b8, located in the domain, not saved (Wensvoort, 1989, J. Gen. Virol., 70: 2865-2876). Enzyme-linked immunosorbent assay with a comprehensive trapping lock is custardy, infected pestiviruses. Thus, with this analysis it is possible to distinguish pigs infected with a strain of the virus of classical swine fever, swine infected with, for example, a strain of the virus of viral diarrhea in cattle. However, this test cannot distinguish pigs infected with wild virus strain of classical swine fever, swine vaccinated With strain. In addition, this analysis is not suitable for use with the subunit vaccine E1, regardless of whether it is live or inactivated.

One test in accordance with the present invention is a modified enzyme-linked immunosorbent assay with a comprehensive trapping-blocking on the basis of a single monoclonal antibodies, for example b3. Such enzyme-linked immunosorbent assay with a comprehensive trapping-blocking on the basis of one monoclonal antibody that is used to bind antigen with the surface of the tablet to perform enzyme-linked immunosorbent assay, and competition with wild whey, still not documented in the scientific literature and is an integral part of the present invention. Now, when a well-known Finance other antibodies, including antibodies against other viruses or other diseases, or antibodies, which are indicators of other conditions of the human or animal. Therefore, this discovery is useful for all enzyme-linked immunosorbent assays or similar assays using complex trapping-blocking, which are developed on the basis of a single monoclonal antibodies and dimenisonal or multisensornogo antigen. Presented in the application test method is applicable also to identify other members of the pairs of molecules binding partners, such as activators/receptors, enzymes/inhibitors, and others, in which one partner has at least two identical binding site.

Thus, this invention also provides a method of determining the presence of a test substance (e.g., antibody) capable of selectively bind with the binding site of the partner (e.g., antigen), on the basis of competition test substance with the measured amount of the reference substance (antibody) capable of selectively bind with the binding site of the partner, which includes

1) contacting the sample with

(a) the reference substance (what ecoloy a partner, containing at least two identical binding site for the reference substance, and

(c) a reference labeled substance (antibody);

2) measurement of the degree of separation of labeled substances from the media.

For example, the binding partner (antigen) for the reference substances (antibodies) that contains at least two identical binding site, is the dimer binding partner (antigen) with the reference substance.

Based on the same principle, the method of determining the presence of a test substance (antibody) having at least two identical binding site in one molecule, which are designed to bind with a binding partner (antibody), which includes:

1) contacting the sample with (a) a binding partner (antibody) associated with a solid carrier, and (b) a labeled binding partner (antibody);

2) measuring the extent of binding of the labeled substance with a carrier.

In these methods, antibodies and antigens are given only as an example; they can be replaced by molecules binding partners.

The present invention further predusmatrivaetsya diagnostic kit containing:

a) reference monoclinic crystal is IDT

(C) antigen complex with a reference antibody that contains at least two identical binding site for the reference antibody, or a complex of components (a) and/or (b) and (C), as well as other components necessary for performing a competitive immunoassay.

This method is suitable for differential diagnostic testing using subunit vaccines E1, in which the removed one or more epitopes of the protein E1, for example, domain A. This test can be used with subunit protein E1, where the domain And was subjected to mutation, in which antibodies are produced against such mutated domain And not compete with monoclonal antibody b3in relation to the epitope of this antibody. In addition, this test can be used with modified C-strain or other vaccine strains of the virus of classical swine fever in which the domain was replaced by the domain pestivirus related to other antigenic group as well as the virus of classical swine fever (see above), or has been subjected to mutation, which resulted in antibodies that affect this domain, not compete with monoclonal antibody b3against this epitope anitanola monoclonal antibodies b8can be used in conjunction with a vaccine, which removed the domains b + C or a domain or domains In + or domain replaced by similar domains pestivirus related to other antigenic group, as the virus of classical swine fever (see above), or domains In + or domain were motivovany so that antibodies produced against these domains do not compete with monoclonal antibody b8in relation to the epitope of this antibody. This test is illustrated on the basis of monoclonal antibodies b3or monoclonal antibodies b8strain Brescia. However, this test can be successfully used with other monoclonal antibodies directed against domain a or domain b + C E1 protein of strain Brescia or against the domain or domains In + any other strain of the virus of classical swine fever, as well as with monoclonal antibodies produced against similar domains in the protein E1 of any other pestivirus. This test can also be performed on the basis of the epitopes of the E2 protein (see example 5). The antigens used in the (modified) enzyme-linked immunosorbent assays with complex trapping lock of the present invention, preferably are dimers or multiparae protein E is Noah fever, interacting with a monoclonal antibody b3or b8or with the same monoclonal antibodies directed against epitopes of the protein E2. In the case of vaccines with the mutated domain And dimers or multimeric antigen used for diagnostic test, can be synthesized by removing structures b + C (see example 5), and in the case of vaccines with the mutated domains b + C, dimers or multimeric antigen used for diagnostic test, can be synthesized by removing structures (cf. Fig. 5 in relation to structures; cf. examples 4 and 5). Diarizonae (or multipersonal) form of the antigen protein E1 is based on the disulfide bridges formed by the cysteine residues in the C-terminal part of the protein E1. This allows a very sensitive immunoassay because the molecule dimenisonal antigen contains two copies of one epitope monoclonal antibody. Thus, one monoclonal antibody can be used for immobilization dimenisonal antigen through a single epitope and labeling dimenisonal antigen through a different epitope. The competition, created by serum antibodies resulting from infection with the wild strain, intellego test for the presence of these antibodies. The present invention also relates to diagnostic kits based on this method, which include antigens for protein E1 or E2, labeled (enzyme and immobilized monoclonal antibody of the same type, affecting the epitope of the protein E1 and E2, as well as other well-known components (tablets, solvents, enzyme substrate, dyes and so on) that perform the immunoassay competitive type.

The vaccine of the present invention contains a nucleotide sequence as such or in the form of a vaccine strain, the vector or the body-master, or polypeptide, as described above, in a quantity sufficient to protect against pestiviruses infection. This vaccine can be multi-purpose and include other immunogenic or nucleotides. In addition, such vaccines may contain known carriers, adjuvants, solvents, emulsifiers, preservatives, etc. Vaccines of the present invention can be obtained by the known methods.

The method according to the present invention, which is designed to produce infectious transcripts of full-length DNA copy of the genome of the strain of the virus of classical swine fever, in particular the C-strain, can be used to with the strain of the virus of classical swine fever, can also be used for in vitro attenuation (modification) C-strain or any other strain of the virus of classical swine fever or pestiviruses strain with the purpose of receiving the vaccine.

The vaccine is based on the C-strain of the present invention allows using serological methods to distinguish vaccinated pigs from pigs infected with the wild strain of the virus of classical swine fever. Vaccine-markers on the basis of any other strain of the virus of classical swine fever or pestiviruses strain with the same degree of reliability to be obtained using the methods of the present invention. Such vaccines markers can be created by mutation (deletion, site-directed mutations, insertions) region that encodes a protein E1, N-terminal part of the protein E1, domains a or b + C E1 protein, a region that encodes a protein E2-strain, similar regions in the genomes of strain or other pestivirus strains, or by replacing these areas with appropriate areas of pestiviruses, including other antigens, or pestiviruses related to other antigenic group.

An alternative way of creating a vaccine-marker based on the C-strain is the inclusion in its genome the heterologous GENETIChESKAYa pigs, or have an artificial origin and not usually found in the body of pigs.

In addition, such heterologous genetic material can encode antigens, indicating protective immunity against diseases caused by pathogenic for swine microorganism. Therefore, the scope of the present invention includes the use of C-strain or strains obtained on its basis, or other pestiviruses strain as a vector designed to expressii heterologous antigens that protect against a specific disease in the body is the master, and the body of the host is a mammal. The structure of the recombinant viruses of strain expressing a heterologous sequence, and sites suitable for the insertion of these heterologous sequences have been described above. Similar recombinant viruses can be created for viruses, derived from the C-strain, or for any other pistiros. Therefore, these viruses are also included in the scope of the present invention.

An integral part of the present invention relates to immunogenic potential subunit E1 protein with deletions in domain a or domain B + C. As shown in table 2, both mutant protein E1 able escia. In the scope of the present invention also includes the use of mutants of the protein E1 with deletions or other mutations in domains a and b + C as an inactivated subunit vaccine or live subunit vaccines, expressed by a vector system in the body of the animal vaccinated against classical swine fever. In addition, the mutated protein E1 together with other antigenic proteins of the virus of classical swine fever, for example E2 protein or its mutant form, can be used as inactivated or live subunit vaccines (see above).

The present invention also includes diagnostic tests designed to distinguish pigs vaccinated with vaccine-marker of classical swine fever virus or subunit vaccine containing (mutated) protein E1 and/or (mutated ) protein E2, from pigs infected with the wild strain pestivirus. Diagnostic testing can be based on serological methods, the detection of antigens or nucleic acids. The choice of test method is suitable for this case, along with other factors depends on the specificity of the used marker. One acceptable form serologicals the Noah trapping, lock, described in example 4. In accordance with the present invention this method using a single antibody is not limited to the detection of the virus of classical swine fever or other pestiviruses and can be used to determine other

antibodies to other diagnostic purposes in humans or animals, as well as to identify other substances with specific binding.

Example of an acceptable test for detection of antigen with the vaccine-marker based on the C-strain is a test that helps detect protein E1 of the wild strain of the virus of classical swine fever and not E1 protein vaccine strain in the blood of pigs. If the domain And the C-strain was replaced by the domain pestiviruses strain relating to other antigenic group compared with the virus of classical swine fever, such testing can be performed on the basis of monoclonal antibodies that recognize conservative epitopes domain And the virus of classical swine fever.

However, if the area of the E2 protein of the C-strain is modified in order to obtain a vaccine token, the recognition of vaccinated and infected animals is made by serology or antigen diagnostike testing can be used as antigen-specific protein sequence E2. The specific sequence of the E2 protein can be obtained from the parent of C-strain (see example 5), from strains of the virus of classical swine fever, which are antigens from C-strain, or from pestiviruses related to other antigenic group compared with the virus of classical swine fever. However, the specific sequence of the E2 protein can also be obtained by mutations (deletions, insertions, or the point heteroplasmy of a mutation) native E2 protein of any pestivirus, however, they can contain (mutated) part of the E2 protein of any pestivirus. As an antigen in diagnostic testing can be used dimeric and multimeric protein E2 (see example 5). In addition, the E2 protein with a monoclonal antibody (compare examples 4 and 5) can be used to perform enzyme-linked immunosorbent assay with complex trapping, lock, main features of which were described above. Diagnostic testing based on the E2 protein described in example 5. If the kit for detection of antibodies is used to detect the protein E2 of pestivirus and based on using a single monoclonal antibodies, this test kit preferably includes an antibody that recognizes rings, diagnostic testing can be performed on the basis of a specific detection modified in the C-strain region of the wild strains of the virus of classical swine fever. Acceptable methods of such testing are hybridization of nucleic acid specific probes and/or amplification by polymerase synthesis reaction chain. The sequence of strain can be distinguished from sequences of the wild strain of the virus of classical swine fever by amplification-based polymerase reaction synthesis circuit (part of) the 3'end non-coding region comprising the sequence TTTTTTTTTTTTT characteristic of the genome of strain.

If a strain is modified by inserting a heterologous sequence token, the scope of the present invention includes any form of diagnostic testing on the basis of this sequence, for example by use of antigen epitopes (epitopes) or histochemical product encoded by this sequence, or by detecting heterologous genetic information using methods of hybridization of nucleic acids, for example by using specific probes and/or amplification, in particular poly is enoma With strain.

Cells and virus. Cells of the kidney pig ( SK6-M, an application for a European patent 351901) were cultured in basic medium Needle containing 5% serum fetal cow and antibiotics. Serum fetal cows were analyzed for the presence of viral diarrhea virus in cattle or antibodies, as described (Moormann and others, 1990, Virology 177: 184-198). Used only serum that does not contain the viral diarrhea virus in cattle and its antibodies. The Chinese vaccine strain (C-strain) of the virus of classical swine fever has adapted to the cells SK6-M as described in the application for the European patent 351901. The strain called "Cedipest" had no cytopathic properties and biologically cloned by three titration endpoint. After three stages of amplification was obtained cloned virus titer equal 3,510650% tissue cytopathogenic dose/ml

The allocation of cytoplasmic RNA from cells SK-6 infected With the strain.

Intracellular RNA from cells infected With a strain that was isolated as described ( Moormann and others, 1990, Virology, 177: 184-198). For this monolayers of cells S6-M in flasks with a volume of 162 cm3(Costar) were infected with strain Cedipest" by several of the TCI were incubated for 1.5 hours at a temperature of 37oWith and added fresh medium to a final volume of approximately 40 ml After 7 hours was added actinomycin D to achieve a final concentration of 1 μg/ml After 24 hours, cells are washed twice with cold phosphate buffered saline and literally ice buffer for lysis (50 mmol Tris-HCl, pH 8,2, of 0.14 mole NaCl, 2 mmole MgCl25 mmol of dithiothreitol, 0.5% (volume ratio) NP-40, 0.5% (weight to volume) desoxycholate sodium and 10 mmol Vanadate-ribonucleoside complexes (New England Biolabs). Lysates were centrifuged (4oC, 5 min, 4000 g ) and for 30 minutes at a temperature of 37oWith supernatant was treated with proteinase K (250 μg/ml, final concentration), and was twice extracted with phenol, chloroform and isoamyl alcohol (49: 49:2) and once was extracted with chloroform and isoamyl alcohol (24:1). RNA was stored in ethanol.

Synthesis and amplification of cDNA

1-2 μg of cytoplasmic RNA from cells infected With the strain, and 20 pmole (-)sense primer were incubated for 10 minutes at room temperature with 1 μl of 10 mmol of methylmercury hydroxide. Denatured RNA was then incubated with 1 ál 286 mmol-mercaptoethanol for 5 minutes at room atoi transcriptase M-MLV, not containing RNase H (Promega), 1 volume of buffer reverse transcriptase M-MLV (50 mmol Tris-HCl, pH 8.3, 75 mmol KCl, 3 mmole gl2and 10 mmol of dithiothreitol) containing 40 units rnasin (Promega) and 80 μl dATP, DSTF, dCTP and dTTP. The final volume of the reaction mixture was 25 μl. The samples were covered with 30 μl of mineral oil (Sigma).

After reverse transcription, samples were denaturiruet for 10 minutes at a temperature of 94oC. Portions of each reverse transcription reaction volume of 2.5 ál of amplified through polymerase chain reaction for 39 cycles (cycle: 94oC, 60 seconds; 55oC, 60 seconds and 72oC, 1-2 min) in 100 µl Taq-polymerase buffer (supplied by manufacturer q polymerase) containing 1 mmol (+) and (-) sense primer, 200 mcmole each of the four nucleoside 5'-triphosphates and 2.5 units q-DNA polymerase (Boehringer Mannheim). The samples were coated with 75 μl of mineral oil (Sigma).

Cloning of a cDNA covering the primary genome-strain

The genome of strain cloned in the fulfilment of the two independent cycles. During the first cycle of the clone (Fig. 1A) primers for the synthesis of single-stranded cDNA and polymerase chain reaction were chosen on the basis of th is of irusa classical swine fever and strains Osloss ( Renard and others, European patent 0208672) viral diarrhea virus in cattle and NADL (Collett and others, 1988, Virorlogy, 165: 191-199). For optimal amplification of the size of the cDNA fragments was chosen equal to 0.5 to 2.5 thousand base pairs. The products of amplification, gel purified, and treated with DNA polymerase, T4 DNA polymerase, 1 maple, and then fosforilirovanii polynucleotides T4. Thereafter, the cDNA fragments ligated using T4 ligase into the site Sml. pGEM4 Z-blue.

In the second cycle of the clone (Fig. 1B), the primers were chosen from the sequences of cDNA clones obtained after the first cycle of cloning. Primers possibly contained the restriction sites suitable for cloning of the amplified cDNA fragments. After reverse transcription and amplification by polymerase chain reaction (see above) cDNA fragments or cut with two different restriction enzymes, or diphosphorous and fosforilirovanii (as described above) at one end and hydrolyzed in an acceptable restriction enzyme at the other end. If it was not possible to use primers restriction sites introduced by polymerase chain reaction, cloning chose the site in amplificatoare FR is ini (Promega) or pGEM5Zf (+) (Promega), hydrolyzed restriction enzymes, forming the ends, comparable to the ends of PCR products.

To obtain cDNA clones containing the last 5'-and 3'-end of the genome of strain, we used a method of ligating the 3'-5'- ends (Mandl and others, 1991, Journal of Virology 65: 4070-4077). Cytoplasmic RNA was isolated from cells infected With the strain, as described above, and purified through a layer of 5.7 sl (Moormann and Hulst, 1988, Virus Res., 11: 281-291). Based on the results, suggesting that the 5'-end of the viral genome viral diarrhea in cattle missing 5'-cap (Brock and others, 1992, J. Virol. Meth., 38: 39-46), the genomic RNA of the C-strain ligated without pre-treatment with pyrophosphatase. 8 µg RNA ligated in a reaction mixture containing 50 mmol Tris-HCl, pH 8.0, 10 mmol gCl210 mmol of dithiothreitol, 20 units rnasin (Promega), 10 μg/ml bovine serum albumin (without RNase) and 1 mmol of ATP, using 10 units of ligase, T4 RNA (New England Biolabs). This mixture is incubated for 4 hours at a temperature of 37oC. RNA was extracted with a mixture of phenol and chloroform, precipitated with ethanol, collected sediment and re-suspended in water, not containing RNase. Portions RNA (2 µg) were back-transcribieron amplified using primer set. For reverse transcription were used (-)sense primer, which was hybridisable 5'end non-coding region. For two stages of amplification in accordance with the polymerase chain reaction, we used the (+) sense primers, hybridities in the 3'end non-coding region, and (-) sense primers, hybridities 5'end non-coding region. After extraction with a mixture of phenol and chloroform and precipitation with ethanol, the products obtained by polymerase chain reaction, hydrolyzed Ncol (included in (+)sense primer used in polymerase chain reaction (PCR) and EAD 1 (nucleotide 81 in sequence with ID 1) and ligated into the Ncol sites-Eaql region U21 (Vieira and Messing, 1991, Gene, 199: 189-194).

All procedures modification and cloning used in example 1 were performed as described (Sam-brook and others, 1989, Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold spring Harbor, PCs, new York. Restriction enzymes and modifying DNA enzymes were bought and used in accordance with the instructions of the manufacturers. Plasmids transformed and maintained in the strain D5 Escherichia coli (Hanahan., 1985, DNA cloning 1: 109-135).

Sequencing of cDNA clones

Plasmid DNA used zentrifugenbau in the caesium chloride (Sambrook and others, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold spring Harbor, PCs, new York. Kit for sequencing on the basis of the T7 polymerase (Pharmacia) was used for direct double-strand sequenced plasmid DNA. In addition S6, T7 and universal forward and reverse primers pUC/M13 used oligonucleotide priming on the basis of the sequence of strain Brescia virus classical swine fever (Moormann and others, 1990, Virology, 177: 184-198). The primers were synthesized by using a DNA synthesizer "Cyclone" (New Brunswick Scientific) or synthesizer DNA/RNA 392 (Applied Biosystems). Products of sequencing reactions were analyzed on 6% acrylamide gel containing 8 moles of urea. Data sequencing were analyzed on a Compaq 386 with hardware "Speedreader and software Intelligenetics Inc., Applied Imageing Corp., Geneva, Switzerland) on an Apple Macintosh computer with software MacMollytetra.

Given the possibility of sequencing errors or discrepancies that are caused q polymerase or heterogeneity of RNA-strain, the entire genomic sequence of the cDNA clone C-strain was determined by sequencing at least two cDNA clones obtained after independent polymerase reactions of the synthesis chain. In case of discrepancies dnow sequence by sequencing the third cDNA clone obtained after performing another independent polymerase reaction synthesis circuit (Fig. 1A).

Example 2.

Synthesis of infectious transcripts of primary DNA copy of the genome of strain.

Construction of cDNA-clone pPRKf1c-113. Primary DNA copy of the genomic RNA of the C-strain was created according to the scheme depicted in Fig. 3. First I created two subclone, one of which (pPRc64) contained the cDNA sequence of the 5'-terminal half of the genome (nucleotides 1-5560) and the second (pPRc111) the cDNA sequence of the 3'-terminal half of the genome (nucleotides 5463-12311). Originally created design of primary cDNA clone was analyzed in pGEM4z-blue. However, this approach was unsuccessful due to the instability of the primary insert in the vector. To increase the stability of clones inserts 5'- and 3'-terminal halves of the clones again cloned into a derivative of the vector rock with a small number of copies (Vieira and Messing, 1991. Gene, 100: 189-194), resulting clones were obtained R108 and pPRcl23. At this point, the vector ROC modified by deleting most of restriction sites, multiple cloning site and the sequence of the T7 promoter. The resulting vector, pPRK, which is used for further primary clone contained f1c-113 has the following structure (Fig. 3). Insert plasmids pPRc45 and pPRc46 connected in the Hpal site located at nucleotide position 1249 sequence-strain (sequence ID 1), forming plasmid pPRc49. Insert pPRc49 then joined the insert R44 in the Nsil site located at nucleotide position 3241 (sequence ID 1), forming pPRc63. the 5'-End half of the clone pPRc64 (nucleotides 1-5560, the sequence with ID 1) was created by joining the insert pPRc63 amplified with polymerase chain reaction) fragment cDNA last 5'end of the genomic RNA of the C-strain. Received the 5'end (+)sense primer containing the EcoRl sites and Satl, for which there was a sequence polymerase promoter T7 RNA, and the first 23 nucleotides of the genomic RNA of the C-strain. This promoter and (-)sense primer of the second cycle of cloning used to amplify a cDNA fragment, which is hydrolyzed by EcoRl and hl and cloned with the formation of pPRc63 by hydrolysis EcoRl-Xhol (nucleotide 216 in the sequence with ID 1). And, finally, insert pPRc64 again cloned with the formation of the pPRK by hydrolysis EcoRl-Xhal, which allowed us to obtain pPRc108.

3'-Terminal half of the pPR clone is obtained in the second cycle (pPRc67, 53, 58 and 55), and one clone obtained in the first cycle (R14). Insert R67 and pPRc53 joined the website Nh1 in nucleotide 7778, education pPRc71. Insert pPRc55 and pPRc58 connected in the Apal website, located at nucleotide position 10387, education pPRc65. Insert R65 and R14 then combined in the website ff11 in nucleotide 11717, education pPRc73. Insert pPRc73 connected with insert pPRc71 in the Pstl site in the nucleotide 8675, education pPRc79. Then insert pPRc79 containing the complete 3'end of the cDNA sequence of the C-strain, modified so that you can enter the Srfl site, which after hydrolysis formed an exact 3'end of the cDNA sequence of the C-strain (exact transcription of the 3'-end). With this purpose, synthesized 3'end (-)sense primer containing sites Srfl and Xbal and 18 nucleotides complementary to the 3'-end sequence of the genomic RNA of the C-strain. This primer and (+)sense primer obtained in the first round of cloning was used to amplify a cDNA fragment. This fragment hydrolyzed using sites Sl (nucleotide position 11866, the sequence with ID 1) and Xbal and cloned with the formation of R79, hydrolyzed posedge specific for the C-strain fragment Nl5532 - Xbalmcs vector pPRc111 inserted into the vector pPRc108, hydrolyzed by N15532 - Xbal mcs.

The design of the primary clone pPRK-f1c-133

The primary cDNA clone pPRKf1c-113 in addition to the "silent" nucleotide mutations included five point mutations, which occurred the same substitution of amino acids in the amino acid sequence selected from at least two cDNA clones obtained in the first series. These five point mutations in clone pPRKf1c-113 was replaced by the predominant sequence (2 and 3) by replacing subjected to DNA fragments corresponding DNA fragments containing a predominant sequence.

the 5'-End half of the cDNA clone pPRKc108 with the point heteroplasmy mutation in position nuleotide 4516 modified by replacing the fragment Scal3413-Ncol5532 vector pPRc108 fragment of the vector pPRc124. Clone R124 was obtained by replacement of Pvull fragment 4485-Nhel5065 vector pPRc44 the corresponding fragment of the vector pPRc32 (cf. Fig. 1). New 5'-terminal half of the cDNA clone was designated as pPRcl29.

In order to clone created the 3'-terminal half of the clone by removing the 5'-end portion sequences of the C-strain pPRKf1c-113 from customers Safl this vector (cf. Fig. 3) to the Hpal site at nucleotide position 5509 (the sequence with identifica 10295. The mutation in position 8526 restored over two stages. First, the Apal fragment 8506-PStl 8675 in pPRc53 was replaced by a fragment of pPRc90 with the formation of the vector pPRcl25. Then the Nhel fragment 7378 - Stl 8675 vector pPRcl23 was replaced by a fragment of vector pPRcl25 education pPRcl27.

To restore the three mutations at positions 9002, 10055 and 10205 first modified R58 to delete the site FSpl in this vector. At this stage, we removed the EcoRl fragment mcs-Ndel vector RRs (gap Ndel in G4Z blue), which allowed us to obtain pPRcl26. Plasmid vector pPRcl26 used for recovery of mutations in the provisions of 10055 and 10205 to replace the fragment Sl9975-Apal10387 the corresponding fragment of the vector pPRc96 education R128. The mutation in position 9002 restored by replacing the fragment of Aat 11-Fspl9016 (gap Aat11 in 4z blue) vector RVs fragment Fspl9016 vector pPRc90 education pPRcl30. Finally, the fragment Stl 8675-Apal10387 vector pPRcl27 replaced by the corresponding fragment of the vector pPRcl30, which allowed to obtain the plasmid pPRcl32. All stages of sublimirovanny, during which you are replacing the individual mutations were verified by sequencing.

Primary clone pPRKflc-133 was created by inserting a fragment Ncol 5532 - bal mcs plasmids pPRcl32 in ve is able mutants of strain, contain other antigens can be obtained from clone pPRKf1c-133 by replacing part of the E1 gene of this design is similar to the genome of strain Brescia virus classical swine fever. For this fragment Nhel2443-Affll 2999 vector R129 replaced by the corresponding fragment h6 (Van Rijn and others, 1992), which allowed to obtain the 5'-terminal half of the hybrid clone pPRc139. Primary hybrid clone pPRKf 1C-h6 was obtained by insertion of the fragment Ncol 5532-Xfbal mcs vector pPRcl32 in pPRcl39. After this operation the clone contains antigenic region E1 strain rscia virus classical swine fever with a specific website Bg111.

All procedures modification and cloning presented in example 2 was performed in accordance with the instructions provided in the opposed material (Sambrook and others , 1989, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold spring Harbor, PCs, new York. Restriction enzymes and DNA modification bought in commercial enterprises and used in accordance with the instructions of the manufacturers. Plasmids transformed and maintained in the strain D5 Escheria coli (Hanahan., 1985 in DNA cloning 1: 109-135).

Transcription of RNA in vitro

Plasmid DNA used for RNA transcription in vitro was purified on Qiagen columns (Westburg) in sootvetstvenno were extracted with phenol and chloroform, precipitated with ethanol, dried under vacuum and dissolved in the appropriate volume of water, not containing RNase.

1 μg of linear plasmid DNA was used as template for in vitro transcription. RNA was synthesized for 1 hour at a temperature of 37oWith 100 µl reaction mixtures containing 40 mmol of Tris-HCl, pH 7.5, 6 mmol MgCl2, 2 mmole spermidine, 10 mmol of dithiothreitol, 100 units rnasin (Promega), 0.5 mmole ATP, GTP, TTF, UTP and 170 units of RNA polymerase T7 (Pharmacia). DNA templates were removed by hydrolysis for 15 minutes at a temperature of 37oUsing Gnkazy 1, not containing RNase a (Pharmacia), followed by extraction with phenol and chloroform and precipitation with ethanol. RNA was dissolved, and 20 μl of water, not containing RNase, and produced quantitative determination by measuring in the ultraviolet region of the spectrum at a wavelength of 260 nm.

Transfection of RNA

Mixture for transfection RNA was obtained by careful mixing 50 μl of the solution lipofectin ( Gibco BRL) (10 µg lipofectin in water, not containing RNase) and 50 μl of the RNA solution (1 µg RNA in water, not containing RNase) and incubation of this mixture at room temperature for 15 minutes. For transfection of RNA used subcloned washed with medium Needle, modified by way of Dulbecco. After that, the cells were added to 1 ml of the above medium, and then the mixture for transfection of RNA. After incubation for 16 hours at a temperature of 37oWith this medium was replaced with 2 ml of eagle medium, modified by way of Dulbecco, to which was added 5% serum fetal cows. Incubation was continued for another 3 days at a temperature of 37oC. Then cells were subjected to immune staining using specific monoclonal antibodies to the virus of classical swine fever in the analysis of monolayer of immunoperoxidase in accordance with the description given by Sensorcom and others (Vet. Environ., 1986, 12: 101-108).

Characterization of recombinant virus-strain

The supernatant of transfected cells was transferred to the plates with a hole diameter 35 mm, containing confluent monolayers S6-cells, and incubated for 5 days at a temperature of 37oC. Cells of transfected monolayers were treated with trypsin, diluted in 7.5 times the medium Needle, modified by the method of Dulbecco, and were cultured for 7 days at a temperature of 37oWith in flasks with a volume of 75 cm2(Costar). After that, cell culture virus a temperature of 4oWith and collected the supernatant.

The virus was characterized by analysis of monolayer of immunoperoxidase and restriction analysis of viable fragments amplified by polymerase chain reaction. After infection S6 cells viruses FLc-h6 and F1c-133 monolayers were incubated for 4 days at a temperature of 37oC. Then the monolayers were subjected to immunome staining monoclonal antibodies against conservative (monoclonal antibody b3, domain a) and nonconservative (monoclonal antibody b5 and b6, domains b+C) epitopes of the protein E1 strain Bresci, as well as monoclonal antibodies specific for the C-strain and directed against E1 protein (monoclonal antibody C2) or E2 (monoclonal antibody C5) (Wensvoort G.,1989, Thesis, pages 99-113, Utrecht, the Netherlands). Monolayers S6 cells infected native Brescia virus or native virus-strain, used in this analysis as control samples. The results are shown in table 1 and correspond to the predicted data. Monoclonal antibody b3 recognizes an epitope of the protein E1, conservative strains of the virus of classical swine fever, and therefore detects all strains presented in table 1. Monoclonal entitytag monoclonal antibody C2 does not recognize the protein E1 strain Brescia and therefore interacts only with strains With and F1c-133. Finally, C5 monoclonal antibody does not recognize the protein E2 strain Brescia and therefore interacts with all viruses, is presented in table 1, except for strain Brescia.

The genomic RNA of the virus F1c - h6 should contain specific Bglll site, which is located in the E1 gene (see above). To ensure the availability of this website, cytoplasmic RNA was isolated from S6-cells, infected with recombinant virus F1c-h6 or virus F1c-133, amplified using polymerase chain reaction described above, using primers described by Van Rijn and others (1993, J. Hen. Virol., 74: 2053-2060), and hydrolyzed in the fragment Bg111 Amplificatory fragment from 1091 base pairs F1-h6 was breaking under the action of Bg111 c education fragments consisting of 590 and 501 base pairs, while amplificatory fragment F1c-133 remained intact.

Example 3

Immunization of piglets deletion mutants of the protein E1

Construction and expression of deletion mutants of the protein E1 strain Brescia virus classical swine fever.

As you know, the E1 protein without a transmembrane region of strain Brescia virus classical swine fever expressed by insect cells, causes the protective immune reactions the data item, A and b+C in the N-terminal half of the protein E1, where antibodies are formed, neutralizing the virus of classical swine fever (Wensvoort, 1989, J. Gen. Virol., 70: 2865-2876; Van Rijn and others, 1992, Vet. Environ., 33: 221-230; Van Rijn and others, 1993, J. Gen. Virol., 74:2053-2060). In addition, antibodies that provide a neutralizing effect on domain a and domain b+C, synergistically to neutralize the virus of classical swine fever (Wensvoort, 1989, J. Gen. Virol., 70: 2865-2876). To assess the immunogenicity of mutant proteins with a deletion of the E1 domain+or domain And were created appropriate constructs in the baculovirus vector and expressed mutant proteins were tested on pigs.

Baculoviruses expressing mutant proteins E1, created by overlap recombination DNA virus NV wild-type (nuclear polyhedrosis virus of Autographa Californica ) and DNA vector transfer rsmo comprising a sequence encoding a specific mutant protein El. Vector transfer rsmo received from s3 (Vlak and others, 1990, Virology, 179: 312-320) by introducing a fragment of T directly in the 5'-end of the first base (G) specific BamH1 site of the last vector. Thus was created the start codon ATA overlying the first Foundation G site Wamn. Messenger RNA was transcribable of heterologous II mutant proteins E1, received from the insert pPRb2 protein E1 (Van Rijn and others, 1992, Vet. Environ., 33: 221-230) by amplification using the polymerase reaction synthesis circuit. In this regard, two primer comprising the BamH1 site in its sequence. 5'End (+)sense primer has the sequence (sequence ID 2). The underlined sequence in the primer are identical to the nucleotides 2362-2381 in the sequence of strain Brescia (Moormann and others, 1990, Virology, 177: 184-198), bold marked the BamH1 site. the 3'End (-)sense primer includes a stop codon, located near the site Wamn. He has the sequence (sequence ID 3). The underlined sequence in the primer corresponds to nucleotides 3433-3453 in the sequence of strain Brescia (Moormann and others, 1990, Virology, 177: 184-198); bold font shows the site Wamn, and italic is a stop codon.

Amplificatoare sequence cloned in the website UMN vector rsmo and checked against the correct orientation in the vector using restriction analysis. The correct vector transfer was identified as pPAb11. Overlapping recombination between virus DNA NV and DNA vector pPAb11, selection and purification of baculovirus vector, expresse., 1993, Virol., 67: 5435-5442). Further study of the protein by El radioimmunoprecipitation analyses and specific monoclonal antibodies E1 was also described Alstom and others (J. Virol., 1993, 67: 5435-5442). The obtained recombinant baculovirus expresses E1 protein of strain Brescia wild-type without the transmembrane region (cf. the second column from the top in Fig. 3). Protein E1 without the transmembrane region is secreted by cells (Hulst and others, 1993, J. Virol., 67: 5435-5442).

The deletion of the region encoding the domains b+C, of the gene of the protein E1 vector pPAb11 produced by replacing Nhel fragment-Bglll in this design the corresponding fragment of the vector h14 (Van Rijn and others, 1993, J. Gen. Virol., 74: 2053-2060). The resulting vector transfer was identified as b16. It included a deletion in the gene of the protein E1, spanning codons 693-746. Similarly, a region encoding a domain And were removed from the vector pPAbll by replacing Nhel fragment-Bg111 vector pPAbll the corresponding fragment of the vector pPEh18 (Van Rijn and others, 1993, J. Gen. Virol., 74: 2053-2060), resulting received vector transfer pPAbl2. This vector includes a deletion in the gene of the protein E1, spanning codons 800-864.

Recombinant baculoviruses expressing the deleted proteins E1, created, selected and characterized in relation to productory of the four (or two) of piglets at the age of 6-8 weeks without evidence of infection with specific pathogens, was vaccinated intramuscularly in the zero (0) day 1 ml of water-oil emulsion containing 4 μg (mutant) protein E1, and re-vaccinated at the 28th day, 1 ml of oil-water double emulsion containing 15 µg (mutant) protein El (table 2). Construction of mutant E1 protein containing a deletion in domain a or domain b/C, and E1 protein wild type described above and represented by the structures shown in Fig. 5. For the first vaccine produced in a zero (0) day, used the supernatant of insect cells infected with the appropriate recombinant baculoviruses. The number of E1 protein in the supernatant was calibrated as described in contrasted to the material (Hulst and others, 1993, J. Virol. , 67: 5435-5442). For powtorki vaccination produced a 28-day E1 protein was purified by immunoaffinity electrophoresis of the supernatant of infected insect cells (Hulst and others, 1993, ibid.). Piglets from all vaccinated groups and unvaccinated control group, which included two animals without signs of infection by specific pathogens, were subjected to control infection by introducing into the nose 100 doses causing death of 50% of organisms strain Brescia 456610 virus classic swing piglets acute form of the disease, which is accompanied by severe fever and thrombocytopenia, occurs on the third to fifth day and becomes a cause of death on the seventh-eleventh day. Samples of heparinized (ethylenediaminetetraacetic acid) took 40, 42, 45, 47, 49, 51, 53 and 56 days post-vaccination and analyzed for the content of platelets and the virus of classical swine fever in accordance with the description given in the opposed material (Hulst and others, 1993, ibid.). Serum samples blood was taken at 0, 21, 28, 42 and 56 day and analyzed by enzyme-linked immunosorbent assay with a comprehensive trapping-blocking (Wensvoort and others , 1988, Vet. Environ., 17: 129-140) and neutralizing peroxidase-linked assay (NLA, Terpstra and others, 1984, Vet. Environ., 9: 113-120) to detect (neutralizing) antibodies against the virus of classical swine fever. The results of testing using enzyme-linked immunosorbent assay with a comprehensive trapping-blocking were expressed as percentage of ingibirovaniya standard signal; inhibition <30% is negative, the inhibition of 30-50% unspecified, inhibition > 50% positive. The titles on the basis of neutralizing peroxidase-linked analysis expressed as the equivalent time the crops.

All animals were daily examined in order to identify symptoms and measured their body temperature. Clinical symptoms were fever, anorexia, leukopenia, thrombocytopenia, and paralysis.

Example 4.

Development of enzyme-linked immunosorbent assay with a comprehensive trapping-blocking (differential enzyme-linked immunosorbent assay with a comprehensive trapping-blocking) for the detection of classical swine fever virus based on a monoclonal antibody

Description diagnostic test.

This example describes enzyme-linked immunosorbent assay with a comprehensive trapping-blocking (CTB-ELIS) and its kind of CTB-DIF (differential enzyme-linked immunosorbent assay with a comprehensive trapping-blocking), which represent a modification of an existing enzyme-linked immunosorbent assay with a comprehensive trapping-blocking (Wensvoort and others , 1988, Vet. Environ., 17: 129-140) and are designed to detect specific antibodies to the virus of classical swine fever.

Differentely enzyme-linked immunosorbent assay with a comprehensive trapping-blocking is based on the discovery that glue is really in the environment dimenisonal protein E1. This protein was detected in the analysis of media from cells infected with the above baculovirus, on a Western blot after electrophoresis in polyacrylamide gel with sodium dodecyl sulfate in non conditions. In E1 dimers have two copies of the epitope (one for each monomer) for specific monoklonalnyh antibody protein E1. Thus, together with the dimeric antigen can be used a specific monoclonal antibody specific protein E1, which is immobilized antibody engraved on the walls of the hole tiralongo microplate, and simultaneously detecting antibody linked to horseradish peroxidase.

Differential enzyme-linked immunosorbent assay with a comprehensive trapping-blocking can be applied together with the subunit vaccine E1, which has a deletion in domain a (see Fig. 5 to familiarize yourself with the design). With this method was able to differentiate specific antibodies of classical swine fever virus, resulting in piglets vaccinated with protein-E1 with the remote domain, and specific antibodies to the virus of classical swine fever, resulting in piglets infected slabovrajennami strains Henken, Zoelen, Verdeschi pathogens, which were assigned numbers 766, 786, 789 and 770, were vaccinated with the mutant protein E1, which has a deletion in the domain And, as described in example 3 (see also table 2), and subjected to the control of infection with the virulent strain Brescia virus classical swine fever on the 44th day after vaccination. Produced assays of serum taken at 28, 42 and 56-day.

Serum against slaboperemennykh strains of the virus of classical swine fever was also received in groups of four pigs with no signs of infection with specific pathogens. Serum taken from pigs infected with strains Henken, Zoelen, Vegda and 331 were analyzed at 0, 21, 28 and 42 day after infection. Serum taken from pigs vaccinated with vaccine Cedipest, were analyzed at 0, 44, 72 and 170th day after vaccination.

The above serum was analyzed using three different tests. Test 1 is a neutralizing peroxidase-linked analysis, described Terpstra and others, 1984 (Vet. Environ., 9: 113-120), which is intended for the detection of neutralizing antibodies against the virus of classical swine fever. Test 2 is a solid phase enzyme-linked immunosorbent assay with a comprehensive trapping-blocking (Wensvoort and others, 1988, Vet. Environ., 17: 129-140), prednaznachalos enzyme-linked immunosorbent analysis with complex trapping lock used monoclonal antibody b3 (also known as CV1-HCV -39.5) (Wensvoort 1989, J. Gen. Virol. Virol., 70: 2865-2876) capable of recognizing the epitope in domain A1 E1 protein of the virus of classical swine fever. Wells for enzyme-linked immunosorbent assay was covered with a monoclonal antibody b3 (dilution 1: 2000) (immobilized antibody). After washing the holes in them were injected monoclonal antibody b3, linked to horseradish peroxidase (dilution 1: 4000) (detection antibody). Wednesday from Sf21 cells infected with baculovirus forming E1 with the transmembrane region and containing dimenisonal E1 protein at concentrations up to 20 μg/ml, was diluted in the ratio of 1:500 and pre-incubated in the test serum (dilution 1:2,5). Mix the serum with the antigen was added to the conjugate in the wells for enzyme-linked immunosorbent assay. After incubation, the wells were washed again and added to the solution of the Chromogen in the substrate. If immobilized and conjugated monoclonal antibody binds to the antigen, horseradish peroxidase causes chromogenic reaction, indicating that the test serum is negative for antibodies to the virus of classical swine fever. If this epitope in the antigen is blocked by antibodies test what I'm about that the test serum contains domain A1 antibodies against the virus of classical swine fever. The results obtained during three different serological tests, are given in table 3.

On the 42nd day post-vaccination sera from pigs vaccinated with the E1 protein with a deletion of the domain And responded when performing neutralizing peroxidase-linked analysis and enzyme-linked immunosorbent assay with a comprehensive trapping-blocking and did not react in the process of performing a differential enzyme-linked immunosorbent assay with a comprehensive trapping lock. After control of infection by strain Brescia virus classical swine fever virulent serum of the same pigs were positive in all three tests on the 56th day after vaccination (day 12 after infection control), indicating that after controlling infection occurred booster effect. Beginning with the 21st day after infection, sera from pigs vaccinated with strains Henken, Zoelen, Bergen and 331, respond positively when performing neutralizing peroxidase - linked analysis, enzyme-linked immunosorbent assay with a comprehensive trapping lock and differential tzii the same was true for pigs, vaccinated vaccine strain dipest.

Thus, differential enzyme-linked immunosorbent assay with a comprehensive trapping lock allows you to get the results you need and can be used in conjunction with vaccine-marker with the mutated domain And E1 protein of the virus of classical swine fever, as antibodies against mutated domain do not compete with monoclonal antibody b3 in relation to impact on this epitope monoclonal antibody.

The antigen used in differential

enzyme-linked immunosorbent analysis of complex trapping-blocking is dimenisonal E1 protein without a transmembrane region of strain Brescia wild type depicted in Fig. 5. However dimenisonal E1 protein synthesized from the design with a deletion of the domain b+C" shown in Fig. 5, can also be used as an antigen when performing this test.

Example 5

Comparison of enzyme-linked immunosorbent assays with complex trapping-blocking in respect of the detection of classical swine fever on the basis of proteins E1 and E2

Description of diagnostic tests.

In this example, described is RowKey in example 4 and enzyme-linked immunosorbent assay with a comprehensive trapping-blocking on the basis of the E2 protein of the virus of classical swine fever and compared the sensitivity of these methods with the other three enzyme-linked immunosorbent assays with complex trapping-blocking, designed for the detection of antibodies against E1 protein and neutralizing peroxidase-linked (NPLA) (Terpstra and others, 1984, Vet. Environ., 9: 113-120).

In differential enzyme-linked immunosorbent analysis of complex trapping lock in example 4, referred to as El-Bac-DIF in tables 4-8, use the E1 protein without a transmembrane region, synthesized as antigen in insect cells (SF21 cells). In the modified method E1-You-DIF, called El-Bac-dBC-DIF, is used E1 protein without a transmembrane region, synthesized as antigen in insect cells (SF21 cells) with a deletion of the domain b+C (see Fig. 5). In accordance with Western blot testing E1 protein without a transmembrane region with remote domains In+is secreted from cells in the form of a dimer (results not shown). Testing according to the method of El-bac-dBC - DIF was performed as follows. Wells for enzyme-linked immunosorbent assay was covered with a monoclonal antibody b3 (dilution 1:4000) (immobilized antibody), were incubated for 16 hours at a temperature of 37oC and washed. The medium containing dimenisonal antigen El-dBC with a concentration of 20 μg/ml, was diluted in the ratio of 1: 50 and pre-incubated with testiruyemogo enzyme immunoassay was applied a mixture of serum with the antigen. After incubation for 1 hour at a temperature of 37oWith the wells were washed and added monoclonal antibody b3, conjugated with horseradish peroxidase (dilution 1:1000) (detection antibody). After incubation for 1 hour at a temperature of 37oWith the wells were washed again and added to the solution of the Chromogen in the substrate. Chromogenic reaction was performed for 10 minutes at room temperature. The results of the chromogenic reaction was interpreted the same way as described in example 4.

Other methods enzyme-linked immunosorbent assay with a comprehensive trapping lock, designed for detection of antibodies against E1 protein of the virus of classical swine fever, are presented in tables 4-8, are enzyme-linked immunosorbent assay E1 protein of classical swine fever virus (E1-SFV ELIS) using as antigen a native protein E1 from cells infected with the virus of classical swine fever (Wenvoort and others, 1988, Vet. Environ., 17: 129-140); differential enzyme-linked immunosorbent assays El-You and El-Bac-DIF using the E1 protein without a transmembrane region, synthesized in the form of antigen in insect cells. In enzyme-linked immunosorbent assays rol., 70: 2865-2876) in the form of immobilized and detecting antibodies, while in enzyme-linked immunosorbent analysis El-Bac-DIF is used only monoclonal antibody b3 as immobilized, and the detecting antibody. Enzyme-linked immunosorbent assay El-CSFV carried out precisely in accordance with the description given by Sensorcom and others, 1988 (Vet. Environ., 17: 129-140). Enzyme-linked immunosorbent assays El-Bac and El-Bac-DIF was performed as described above for enzyme-linked immunosorbent assay El-Bac-dBC-DIF when making the following changes. In enzyme-linked immunosorbent analysis El-Bac antigen used was a solution with a ratio of 1:400 dimenisonal E1 protein in the medium of SF21 cells infected with the E1 protein of baculovirus (cf. Fig. 5) at a concentration of 20 μg/ml Monoclonal antibody b8 conjugated to horseradish peroxidase, is a detecting antibody in this modification enzyme-linked immunosorbent assay and is used in the form of a solution with a degree of dilution of 1:1000. In differential enzyme-linked immunosorbent analysis El-Bac-DIF uses the same antigen in enzyme-linked immunosorbent analysis El-Bac, but at the dilution of 1: 200. Monoclonality as the detecting antibody with the degree of dilution of 1:1000.

In enzyme-linked immunosorbent analysis of E2-You used the E2 antigen of the virus of classical swine fever, synthesized in SF21 cells infected with baculovirus SE (Hulst and others, 1994, Virology, 200: 558-565). Because of the infected insect cells is not allocated protein E2, used the lysate of these cells. Like a squirrel E1 a large part of the E2 protein is found as demonizovana molecules, the analysis of lysates of infected cells in adenocarinoma conditions on the gels for electrophoresis in polyacrylamide gel with dietersheim sodium (results not shown). Enzyme-linked immunosorbent assay with a comprehensive trapping-blocking based on this antigen E2 allows you to achieve optimum results when used with a monoclonal antibody C5 and C12 (Wensvoort G., 1989, In Thesis, pages 99-113, Utrecht). However, you can also use protein E2 only with a monoclonal antibody C5 or C12. In the competitive analysis of monoclonal antibodies C5 and C12 inhibit the activity to each other in terms of binding to protein E2. This suggests that these monoclonal antibodies recognize the same or overlapping epitopes in the protein E2 (results not shown). Enzyme-linked immunosorbent assay the tablet for enzyme-linked immunosorbent assay (16 hours at a temperature of 37oC. then the wells were washed. Lysates of SF21 cells infected with baculovirus SE diluted in a ratio of 1:1250, pre-incubated with the test serum (1:1) for 0.5 hours at a temperature of 37oC. In the wells sensitized tablets was administered to mix the serum with the antigen, and incubated for 1 hour at a temperature of 37oC. After that, the tablets were washed and incubated with a monoclonal antibody C5 conjugated with horseradish peroxidase (with a degree of dilution 1:2000). After incubation for 1 hour at a temperature of 37oWith the tablets again washed and added to the solution of the Chromogen in the substrate. Chromogenic reaction was performed for 10 minutes at room temperature. The results of the chromogenic reaction was interpreted the same way as in example 4. All of the above dilutions were made in a buffer for neutralizing peroxidase-linked analysis + 4% of photosystem (Terpstra and others, Vet. Environ., 9: 113-120).

Table 4 shows the results of serum analysis the three little pigs with no signs of infection with specific pathogens, which were vaccinated with vaccine Cedipest using the above enzyme-linked immunosorbent assays with complex trapping-blocking and Nate is e vaccination. In tables 5-8 shows the results of serum analysis group of five piglets without specific pathogens, infected slabovrajennami strains 331, Bergen, Henken and Zoelen of classical swine fever virus, which was performed using the methods described above, enzyme-linked immunosorbent assay with a comprehensive trapping-blocking and analysis based on the neutralizing peroxidase. Serum was analyzed for 0, 10, 14, 17, 24, 28, 35 and the 42nd day after infection. Starting from the 16th day after vaccination, the serum of piglets vaccinated with strain Cedipest responded in each of the five methods enzyme-linked immunosorbent assay, and in neutralizing peroxidase-linked assay. In this period of time sensitivity enzyme-linked immunosorbent assays E2-You and El-Bac-dBC-DIF was as high as can be, and higher than the other three enzyme-linked immunosorbent assays with complex trapping lock. 37-th 170 th day after vaccination all serum alike respond positively in all five enzyme-linked immunosorbent assays, and in neutralizing peroxidase-linked assay. Sera from pigs infected slabovrajennami strains of the virus of classical swine licorice, Rea is tradeswomen peroxidase-linked assay. Except for a few cases, the comparability of the results of the serum in five enzyme-linked immunosorbent assays with complex trapping-blocking and neutralizing peroxidase-linked analysis took place from 21 to 42 days after infection. Had to analyze large quantities of serum of animals infected slabovrajennami strains to conclude whether there are significant differences between the sensitivity reached in five enzyme-linked immunosorbent assays with complex trapping-blocking at an early stage of infection (up to 17-th day).

We can conclude that the enzyme-linked immunosorbent assays E2-You and El-Bac-CTB - DIF are characterized by a high degree of efficiency. Therefore, enzyme-linked immunosorbent assay E2-You can be used in conjunction with vaccine-marker virus classical swine fever (e.g., mutated or neutropenia subunit protein E1, vaccine-marker-strain, modified in the field E2 protein), which does not cause formation of an antibody that competes with monoclonal antibodies in enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assay El-Bac-dBC-DIF is Ermentau analysis with complex trapping-blocking example 4) suitable for use with the vaccine marker virus classical swine fever with the mutated domain And protein E1, since antibodies against mutated domain And do not compete with monoclonal antibody b3 for the epitope for this antibody.

Description of drawings

In Fig. 1 shows a schematic representation of cDNA clones used to determine the nucleotide sequence of the C-strain. In Fig. 1A shows a first series of cDNA clones (see text). cDNA clones with 32 rooms, 90 and 96 used to replace pPRKf1c-113 on pPRKf1c-133 (see example 2). Clone 14 was the only one cDNA clone of the first series, which was used for construction pPRKf1c-113 (see Fig. 3). In Fig. 1B presents the second series of cDNA clones (see text). Numbered cDNA clones of the second series used to create pPRKf1c-113 (see sequence ID 1). The provisions of the cDNA relative to the nucleotide sequence of the genome of strain indicated by a scale ruler (thousands of base pairs) in the lower part of the drawing. Schematic illustration of the identified currently, the genes of the virus of classical swine fever and their patterns in the genome of this virus is given in the upper part of the drawing.

Experts in the field of nomenclature pestivirus proteins do: 1-10), etc. 44/48(Thiel and others, 1991, J. Virol., 65: 4705-4712) or EO (Rumenapf and others, 1993, J. Virol. 67: 3288-3294). Protein E2 is also referred to as Dr (Tamura and others, 1993, Virology, 193: 1-10), gp 33 (Thiel and others, 1991, J. Virol., 65: 4705-4712) or El (Rumenapf and others , 1991, J. Virol. , 67: 3288-3294). Protein E1 of the present invention is also referred to as gp53 (Tamura and others, 1993, Virology, 193: 1-10), gp 55 (Thiel and others , 1991, J. Virol., 65: 4705-4712), etc 51-54 (Moormann and others, 1990,. Virology, 177:184-198) and E3 (Rumenapf and others, 1993, J. Virol., 67:3288-3294). N-terminal autoprotease Nprothe virus of classical swine fever (P20 virus viral diarrhea in cattle, Wiskerchen and others 1991, J. Virol. , 64: 4508-4514), also referred to as R23 is applied, was identified by Tien and others , 1991 (J. Virol., 65: 4705-4712). Cleavage recognition sequence of this protease conserved in pestivirus, leads to the formation of the N-terminal, C-strain (Str and others, 1993, J. Virol., 67: 7088-7095).

In Fig. 2 shows a comparative analysis of the primary structure of the nucleotide sequences 5'-end and 3'-end (In) non-coding regions of strains rsia, Alfort and the virus of classical swine fever. Except for the first 12 nucleotides of the 5'-terminal non-coding sequence of strain Brescia was described by Mormondom and others, 1990, J. Virology, 177: 184-198. The first 12 nucleotides of the 5'end non-coding region of strain Brescia previously not been considered scientific if the ligating the 3' and 5'end of the RNA, described in example 1 of this patent application. Except for the first 9 nucleotides of the 5'-terminal non-coding sequence of strain Alfort was described by Meyer and others, 1989, Virology, 171: 555-567. The first nine nucleotides of the genome of strain Alfort were presented by Meyer in his dissertation, entitled "Virus der Klassischen Schweinepest: Genomanalyse und Vergleich mit dem der Virus Bovinen Viralen Diarrhoe", 1990, tübingen, Germany. Sequence 3'-terminal non-coding regions of strains Brescia and access were described by Mormondom and others, 1990, Virology, 177: 184-198, and Meyer and others, 1989, Virology, 171: 555-567. The start codon ATG and stop codon TGA large open reading frame (cf. the sequence with ID 1) are underlined.

In Fig. 3 gives a schematic design of the primary cDNA clone pPBKflc-113. The numbers of clones are given in the legend to Fig. 1. Sites merge inserts of clones shown by vertical lines. Sites corresponding to these lines are listed in the lower part of the figure. The underlined numbers clones represent cDNA clones with vector sequences ROC (Vieira and Messing, 1991, Gene, 100: 189-194) (see Fig. 4). 5'- and 3'-ends pPRKflc-113 were obtained by extension with amplification of cDNA fragments by polymerase chain reaction (see example 2). Amplificatoare fragment the market of the genome of the virus of classical swine fever has been described in the legend to Fig. 1.

In Fig. 4 gives a schematic representation of vector sequences and primary cDNA inserts in clones Rflc-113, pPRKflc-133 and Rf1-h6. The structure of the vector pPRK derived rock (Vieira and Messing, 1991, Gene, 100: 189-194) described in example 2. CapR, the gene for resistance to kanamycin; ORI, the site of replication initiation; i, the gene that encodes the repressor gene-galactosidase; RO region of the promoter/operator gene-galactosidase; lac Z, part of the gene-galactosidase, encoding subunit-galactosidase. Contains several restriction sites of the vector and the sequences flanking the primary insert in the vector. Appropriate sites were considered in example 2. Pointers and rooms in pPRKflc-113 correspond to the five nucleotides of codons, which were replaced in this design, the result of which was obtained clone pPRKflc-133. The latest design includes the sequence specified in sequence ID 1.

The black square in the pPRKf1-h6 marked area E1 protein of clone Rflc-133, which was replaced with the corresponding region of strain Brescia Are the transcripts derived from a particular primary structure, infectious (+) or not (-), are indicated to the right of this design. T7, posledovatelnyi the nucleotide sequence of the C-strain, specified in sequence ID 1.

In Fig. 5 provides a schematic representation of mutant proteins E1, expressed in insect cells by a baculovirus vector. All proteins E1 encoded by the nucleotide sequence of the strain Brescia (Moormann and others, 1990, Virology, 177: 184-198) and start at the N-Terminus fragment of LyS in position of the codon 668 in a large open reading frame of this sequence. With the end of the native protein E1 is a piece Lu in the position of the codon 1063 in a large open reading frame, while With the ends of the other three proteins E1 are in the position of amino acids 1031. Point framework of marked N-terminal signal sequence covering amino acid residues 668-689, internal hydrophobic sequence covering amino acid residues 806-826, and C-terminal transmembrane region located in the region spanning amino acid residues 1032-1063 protein E1. The deleted amino acid sequences in mutant proteins with a deletion of the E1 domain b+C or As indicated by the gaps in the bars representing these proteins. The location of these deletions relative to the amino acid sequence of E1 protein can be identified by large-scale inalmost, coded a large open reading frame strain Brescia.

1. The nucleotide sequence corresponding to the genome of the virus of classical swine fever (CSFV), characterized in that it includes encoding part of the nucleotide sequence of CSFV strain shown in SEQ ID NO: 1 or a complementary sequence, or RNA equivalent of such nucleotide sequence.

2. The nucleotide sequence corresponding to the genome of the virus of classical swine fever (CSFV), comprising the nucleotide sequence encoding the amino acid sequence 268-494 amino acid sequence shown in the sequence SEQ ID NO: 1 or a complementary sequence, or RNA equivalent of such a nucleotide sequence and/or nucleotide sequence that carries a mutation in the nucleotide sequence that encodes the amino acid 690-877, the amino acid sequence shown in the sequence SEQ ID NO: 1 or a complementary sequence, or RNA equivalent of such nucleotide sequence, and said mutation is selected from deletions or substitutions of amino acids 1-188 in the sequence 690-877, substitutions one is adequate part of the genome of another strain of pestivirus, insertion of a heterologous nucleotide sequence.

3. The nucleotide sequence under item 2, characterized in that it contains an insertion of a heterologous sequence between amino acids 690 and 691 or 691 and 692 in the amino acid sequence of SEQ ID NO: 1.

4. The nucleotide sequence corresponding to the genome of the virus of classical swine fever (CSFV), comprising a nucleotide sequence encoding at least the amino acid sequence 690-1063 SEQ ID NO: 1 or a complementary sequence, or RNA equivalent of such nucleotide sequences, and optionally containing a mutation in the sequence that encodes the amino acid sequence of 268-494 SEQ ID NO: 1, and the mutation is selected from deletions or substitutions of one or more amino acids, altering at least one epitope in the sequence 268-494, substitutions of one or more amino acids encoded by this nucleotide sequence, amino acids, the encoded portion of the genome of another pestiviruses strain, insertion of a heterologous nucleotide sequence.

5. The nucleotide sequence under item 4, wherein the sequence SEQ ID NO: 1.

6. The nucleotide sequence encoding pestiviruses polypeptide corresponding to the amino acid sequence 690-1063 SEQ ID NO: 1, characterized in that it contains a mutation in one of the epitopes in the amino acid sequence 691-750 or 785-870, and this mutation changes mentioned epitope.

7. The nucleotide sequence under item 6, characterized in that it contains an insertion of a heterologous sequence between amino acids 690 and 691 or 691 and 692 in the amino acid sequence of SEQ ID NO: 1.

8. The polypeptide of the virus of classical swine fever encoded by a nucleotide sequence according to any one of paragraphs. 1, 2, 4 or 6.

9. The nucleotide sequence of the strain of the virus of classical swine fever, suitable as vaccines, which is a full-size DNA copy or infectious transcript nucleotide sequence according to any one of paragraphs. 1, 2, 4 or 6.

10. Pestivirus vaccine against the virus of classical swine fever containing polynucleotide having a sequence according to any one of paragraphs. 1-7, or the polypeptide under item 8, or the nucleotide sequence of the vaccine strain under item 9, in a quantity sufficient to protect against virus, what about at least the nucleotide sequence encoding the antigenic pestiviruses polypeptide, antigenic pestiviruses polypeptide and/or antibody induced against antigenic pestiviruses polypeptide, wherein the nucleotide sequence has a sequence according to any one of paragraphs. 1-7, the polypeptide is a polypeptide under item 8, or antibody induced against the polypeptide under item 8.

12. Diagnostic method to differentiate animals infected with pestiviruses from vaccinated animals, and these vaccinated animals were vaccinated pestiviruses the polypeptide or strain pestivirus containing a mutation in the amino acid sequence 268-494 or 690-1063 in SEQ ID NO: 1 which includes contacting the test sample with pestivirus antigen corresponding to amino acid sequence 268-494 or 690-1063 or parts thereof, with an antibody directed against an epitope specified pestiviruses antigen, the epitope is not functional in the mutated polypeptide or pestiviruses the strain used for vaccination, and measuring the degree of competition between the antibody in the above-mentioned sample and antibody directed prot is dimeric or multipersonal the polypeptide and some of the listed antibodies immobilized, and the other part of the antibody machina.

14. The method according to p. 12 or 13, characterized in that pestiviruses polypeptide and the antigen correspond to the sequence of amino acids 690-1063 and the specified epitope is located between amino acids and 870 785.

 

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