Recombinant plasmid pci-neo-odc-nsp1 and its variants pci-neo-odc-e2-l and pci-neo-odc-e2-m encoding venezuelan equine encephalomyelitis (vee) virus proteins and ornithine decarboxylase protein designated for development of agents for prophylaxis of viral infectious diseases (with vee virus example)

FIELD: genetic engineering, biochemistry, virology.

SUBSTANCE: invention proposes recombinant plasmid pCI-neo-ODC-nsP1 and its variants pCI-neo-ODC-E2-L and pCI-neo-ODC-E2-M encoding proteins of Venezuelan equine encephalomyelitis (VEE) and ornithine decarboxylase protein. Plasmids are prepared by the sequential cloning gene-equivalent of non-structural protein nsP1 (fragments of L,M of the structural protein E2) of VEE virus as component of gene encoding enzyme ornithine decarboxylase being firstly into prokaryotic plasmid vector pET-23b and then into eucaryotic plasmid vector pCI-neo. Prepared plasmids can be used in the development of protective preparations used against Venezuelan equine encephalomyelitis virus as a prophylactic agent with respect of this pathogen.

EFFECT: valuable biological and medicinal properties of plasmid and agents.

3 cl, 2 tbl, 6 dwg

 

The invention relates to the field of molecular biology.

The invention can be used in virological, serological, immunological and molecular biological research on the development of protective the next generation of drugs against viral infectious diseases.

The problem of specific prophylaxis of viral infections is one of the most important, because in the structure of morbidity and mortality in the population of the Russian Federation these diseases are one of the leading places. Currently this issue is important due to the low antiviral efficacy widespread currently chemotherapeutic drugs.

Advances in modern molecular biology, genetic engineering and biotechnology discover new and promising directions in the field of creation of effective vaccines of different classes.

The new generation vaccines include DNA vaccines, recombinant vector vaccines, peptide vaccines, transgenic vaccines and antiidiotypic vaccines [1]. Parallel to the development of methods for designing vaccines they receive new information on the mechanisms for the presentation of antigens to the immune system of the host. So, now formed the view that cell organelles animal proteasome - involved in processing of antigens presented by molecules of the first class major histocompatibility complex. Implemented this process in close connection with ubiquitously system [2].

Addition of ubiquitin potential specificity of the proteasome complex has a number of other constitutive peptides mammals that are associated with the presence in their sequences of combinations of certain amino acid residues, which is a strong signal degradation (degradome). The latter applies to the enzyme ornithindecarboxilase (ODC), which is currently selected and cloned in several plasmid vectors [3]. Using interdiscursivity as leader of the protein upon receipt of hybrid proteins may allow transport of any of the target peptide in the proteasome complex, which will lead to his proteasomedependent degradation. The transfer of this mechanism on the construction of new generation vaccines could lead to a highly antigenic preparations devoid of additional "background" loads, leading to various kinds of post-vaccination complications. To implement the above approach can, for example, if proteasomedependent degradation of targeted chimeric antigen transfer in vivo. In this case, polonious hybrid gene in the vector is Oh plasmid under a strong eukaryotic promoter, you can use DNA vaccination.

One of the most effective eukaryotic plasmid vectors is a vector pCl-neo (Fig 1)related to the type of Fig. The website ori used in the plasmid pUC-type, provides a high yield of plasmid DNA. Vaccine plasmids, created on the basis of Fig produce up to 30 μg/ml of plasmid DNA under cultivation on rich liquid nutrient media. Thanks gene resistance to antibiotics only plasmodiidae bacteria able to grow on media containing the antibiotic.

To achieve efficient expression in humans using such regulatory elements, as early promoter of cytomegalovirus (CMV) human promoter of the rous sarcoma virus, the early promoter of SV40 virus.

Also in plasmid injected transcription termination sequence, which typically use a 3'-noncoding poly-A region of the gene of bovine growth hormone or the termination sequence of the SV40 virus.

The efficiency of expression of many genes in eukaryotic cells depends on the presence of plasmid intron, which typically use intron a [4, 5].

As a model of infectious diseases against which developed a preventive tool, choose a well-studied pathogen.

Virus Venezuela the ski encephalomyelitis of horses (VAL) is one of the most fully studied as structural, and from the point of view of the immuno - pathogenesis causative agents of viral infections. Structurally, the causative agent contains a single RNA molecule, covalently associated with a protein capsid surrounded by a dimer of membrane glycoproteins E1 and E2, however, in conditions of high multiplicity of infection is the formation of polyploid virions containing one supercapacitor several nucleocapsids. Replication of the viral genome are transcribed type 2 mRNA - 42S and 26S, defining, respectively, the synthesis of non-structural (nsP), and structural proteins.

Both glycoprotein envelope (E1 and E2) are important for maintaining the infectivity and pathogenicity of the virus, however, if E1 largely determines the phase of the binding of the virus to receptors on the cell membrane and subsequent mirapexin, E2 predominantly induces the body's immune response to viral infection, as shown in studies with serum of convalescents. In this regard, to obtain immunogenic preparations of the virus VAL was selected structural protein of the pathogen E2.

When cloning fragments of structural peptides viruses with antigenic determinants, a necessary condition for their immunogenicity is the preservation of the most relevant epitopes. To date, the antigenic structure of glycoprotein E2 has been well studied, and EP is tops on its surface mapped as an American, and domestic experts [6]. Immunodominant position is highly conservative area, which includes a fragment of the gene of the protein E2 - M virus VAL (figure 2), located in the Central zone E2 protein at a small distance from the site of glycosylation. Less importance is calculated conformational epitopes located in the end regions L and N peptide.

Known recombinant strain of vaccinia virus expressing the proteins of the virus Venezuelan encephalomyelitis of horses (patent (19) RU (11) 2091489 (51)C 12 N 007/00, C 12 N 015/33, C 12 N 015/86, (21) 93018105 (22) 27.09.1997 (71)(73) State research center of Virology and biotechnology "Vector" (54) Strain recombinant vaccinia virus expressing the structural proteins of the virus Venezuelan encephalomyelitis of horses and suitable for the production of immunobiological preparations and how to obtain it).

This invention is the closest analog of the claimed invention for its intended purpose, other characteristics of the present invention has no common features with the previously presented by the State scientific center of Virology and biotechnology "Vector". The strain was obtained by embedding in gene timedancing commercial strain of vaccinia virus (LIWP) under control of the promoter of the protein 7,5 To the vaccinia virus posledovatelno and, encoding structural proteins of the virus VAL (full-size DNA copy of the entire 26S RNA). The result has been a strain of vaccinia virus, which generated the most complete immune response against the virus VAL. The infection of cells obtained by the strain on their surface expressed glycoproteins supercasino shell of the virus Venezuelan encephalomyelitis of horses. To date, however, this invention is not spread in the prevention VAL.

The aim of the present invention to provide a recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M)designed to develop protective the next generation of drugs against the virus Venezuelan encephalomyelitis of horses as a means of prevention against this pathogen.

The essence of the invention is that in the sequential cloning of the gene-equivalent nonstructural protein nsP1 (fragments L, M structural protein E2) virus VAL in the composition of the gene interdiscursivity first prokaryotic plasmid vector pet-23b, and then in eukaryotic plasmid vector pCI-neo in the genetic engineering research obtained recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M). The obtained recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCIneo-ODC-E2-M) has biological characteristics, allows you to use this design as immunobiological drugs (vaccines) and to evaluate its effectiveness.

The obtained recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) has the following characteristics.

1. Pedigree recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) is shown in figure 3.

2. The number of passages by the time certification 1.

3. Standard growing conditions in vitro

The cultivation conditions - culture of E. coli DH5α, HB10 or Jm109 on LB medium with the addition of 25 μg/ml of ampicillin at temperature (37,0±0,5)°C.

4. Molecular-biological properties of the recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M)

Size: 7321 (options 7294, 7291) base pairs (BP)

Marker characteristic: resistance to ampicillin

Data restrictor analysis: see schematic map (figure 4, 5, 6)

Vector part:

The size of the vector part: 5472 base pair

Structural elements: ori-site, the promoter of T7 phage, ori pBR322, the gene of resistance to ampicillin, multiple cloning site, enhancer-promoter of cytomegalovirus and poly (A)sequence of the SV40 virus, a marker gene for resistance to the antibiotic G-418 under the promoter and enhancer of SV40 virus, containing the 3'-end of the synthetic poly (A)sequence.

The cloned fragments is:

Description fragments:

cDNA fragment of the genome of the virus VAL (48-497) (nsP1) and cDNA gene interdiscursivity (figure 4);

cDNA fragment of the genome of the virus VAL (8564-8986) (E2-L) and cDNA gene interdiscursivity (figure 5);

cDNA fragment of the genome of the virus VAL (8990-9409) (E2-M) and cDNA gene interdiscursivity (6).

The size of fragments:

450 BP fragment of cDNA of the gene of viral protein nsP1, 1848 BP - total fragment size ODC and nsP1;

423 BP - fragment of the cDNA of the gene of the viral protein E2-L, 1821 BP - total fragment size ODC and E2-L;

420 BP fragment of cDNA of the gene of the viral protein E2-M, 1818 BP - total fragment size ODC and E2)

Website cloning:

ODC sites NheI-SaI 1; cDNA fragments of genes of the virus VAL on BstXI site, located between the two plots PEST gene equivalents ODC.

5. Stability of recombinant plasmids

Storage for 2 years (time of observation) in the culture of E. coli with the addition of 50% glycerol at a temperature of minus (20,0±2,0)°C.

6. Control of contamination of the culture of recombinant plasmids

Fungal and heterologous bacterial microflora in the culture of E. coli containing a recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) is missing.

7. For more information about recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) are presented in table 1.

8. Brief description of drawings

On Phi is .1 shows the physical map of plasmid vector pCI-neo.

Figure 2 presents the diagram of conformational epitopes of the E2 protein of the virus VAL projected on the cloned fragments of the peptide in the composition of the vector plasmid pet-23b and pCl-neo.

Figure 3 presents the genealogy of recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M).

Figure 4 (5, 6) presents a schematic map data restrictor analysis of recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M).

The best example of the use of recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) can be used as a basis for designing a DNA vaccine against the virus of Venezuelan encephalomyelitis of horses.

An example of executing

A test tube with a culture of E. coli containing a recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) remove from the refrigerator, storage temperature minus (20,0±2,0)°C. carry out cultivation in LB medium with the addition of 25 μg/ml of ampicillin. Duration of cultivation is from 12 to 18 hours at a temperature of (37,0±0,5)°C.

Lysis of bacterial cells carry lysozyme (10 mg/ml in 0.25 M Tris-HCl, pH 8.0) in the presence of 10%SDS. Chromosomal DNA is removed by a high speed centrifugation, followed by a double extraction of nucleic acids with a mixture of phenol/chloroform and one extraction with chloroform. the action of the RNA is removed by gel filtration on columns with separate 2B and precipitate a double volume of distilled cold 70%ethanol. The concentration of the selected plasmid DNA determined spectrophotometrically, given the fact that one of the optical unit at 260 nm corresponds to 50 ág DNA in 1 ml [7]. After sedimentation by centrifugation of plasmid DNA dissolved in THE buffer to a concentration of 1 mg/ml

Isolated and purified DNA preparations appreciate then electrophoresis in 0.8% of agarose gel. Electrophoresis is carried out at a temperature (20,0±2,0)°C for 18 hours when the electric field intensity In 1-2/see plate Size 20×20 cm2.

The resulting product is a purified preparation of recombinant DNA plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M), which can be used as DNA vaccines against the virus Venezuelan encephalomyelitis of horses (table 2).

Recombinant plasmid pCI-neo-ODC-nsP1 (options pCI-neo-ODC-E2-L, pCI-neo-ODC-E2-M) deposited in the special collection of recombinant plasmids Virology centre, Institute of Microbiology of the Ministry of defense of the Russian Federation No. 02/1 (02/2, 02/3).

LIST of USED SOURCES

1. Donnelly J.J., Liu, M.A., J.B. Ulmer, Antigen presentation and DNA vaccines // Amer. J. Respir. Crit Care Med. - 2000. - Vol.162. - P.190-193.

2. Ciechanover A. The ubiqutin-proteasome pathway: on protein death and cell life // J. EMBO. - 1998. - Vol.17. - P.7151-7160.

3. Murakami Y, Matsufuji, S., Kameji So, Hayahi S., Igarashi K., Tamura T., Tanaka K, Ichihara A. Omithine decarboxylase is degraded by the 26S proteasome without ubiquitination // Nature. - 1992. - Vol.360. - P.597-599.

4. Molecular Cloning. (A Laboratory Manual). Ed. by Sambrook J., Fritseh E.F., Maniatis T. Cold Spring Harbor Laboratory Press, 1989. - 1678 p.

5. The website "guidelines on genetic engineering (USA) http://www.molbio.net/.

7. Fields Virology. Edited by B.N. Fields, D.M. Knipe, P.M. Howley et al. Philadelphia, Lippincott-Raven Publishers. - 1996. - 480 p.

6. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular cloning / Lane. from English. edited Bev, Kgosana. - M.: Mir, 1984. - 479 C.

7. Genes B.C. Some simple methods cybernetic data processing diagnostic and physiological studies. - M.: Nauka, 1998. - 208 S.

1. Recombinant plasmid pCI-neo-ODC-nsP1 shown in Fig. 4, encoding a nonstructural protein nsP1 virus Venezuelan encephalomyelitis of horses (VAL), deposited in the special collection of recombinant plasmids Virology centre, Institute of Microbiology of the Ministry of defense of the Russian Federation under No. 02/1, characterized by the following design characteristics:

size 7321 base pairs (BP);

marker characteristic: resistance to ampicillin;

the size of the vector part: 5472 base pair;

structural elements vector part: ori-site, the promoter of T7 phage, ori pBR322, the gene of resistance to ampicillin, multiple site konyrov the tion, enhancer-promoter of cytomegalovirus and poly(A)sequence of the SV40 virus, a marker gene for resistance to the antibiotic G-418 under the promoter and enhancer of SV40 virus, containing the 3'-end of the synthetic poly(A)sequence;

description of the cloned fragment cDNA fragment of the genome of the virus VAL (48-497) (nsP1) and cDNA gene interdiscursivity (ODC);

fragment size: 450 BP fragment of cDNA of the gene of viral protein nsP1;

website cloning: ODC for sites NheI-SaI 1; cDNA fragment of the gene of the virus VAL on BstXI site, located between the two plots PEST gene equivalents ODC;

and intended for the development of DNA vaccines against the virus VAL.

2. Recombinant plasmid pCI-neo-ODC-E2-L, shown in Fig. 5, the coding fragment L structural protein E2 virus, Venezuelan encephalomyelitis of horses, deposited in the special collection of recombinant plasmids Virology centre, Institute of Microbiology of the Ministry of defense of the Russian Federation under No. 02/2, characterized by the following design characteristics:

size 7294 base pairs (BP);

marker characteristic: resistance to ampicillin;

the size of the vector part: 5472 base pair;

structural elements vector part: ori-site, the promoter of T7 phage, ori pBR322, the gene of resistance to ampicillin, multiple SAI is cloning, enhancer-promoter of cytomegalovirus and poly(A)sequence of the SV40 virus, a marker gene for resistance to the antibiotic G-418 under the promoter and enhancer of SV40 virus, containing the 3'-end of the synthetic poly(A)sequence;

description of the cloned fragment cDNA fragment of the genome of the virus VAL (8564-8986) (E2-L) and cDNA gene (ODC);

fragment size: 423 BP - fragment of the cDNA of the gene of the viral protein E2-L;

website cloning: ODC for sites NheI-SaI 1; cDNA fragment of the gene of the virus VAL on BstXI site, located between the two plots PEST gene equivalents ODC;

and intended for the development of DNA vaccines against the virus VAL.

3. Recombinant plasmid pCI-neo-ODC-E2-M shown in Fig. 5, the coding fragment M structural protein E2 virus, Venezuelan encephalomyelitis of horses, deposited in the special collection of recombinant plasmids Virology centre, Institute of Microbiology of the Ministry of defense of the Russian Federation under No. 02/3, characterized by the following design characteristics:

size 7291 base pairs (BP);

marker characteristic: resistance to ampicillin;

the size of the vector part: 5472 base pair;

structural elements vector part: ori-site, the promoter of T7 phage, ori pBR322, the gene of resistance to ampicillin, multiple cloning site, enhe the sør-cytomegalovirus promoter and poly(A)sequence of the SV40 virus, marker gene for resistance to the antibiotic G-418 under the promoter and enhancer of SV40 virus, containing the 3'-end of the synthetic poly(A)sequence;

description of the cloned fragment cDNA fragment of the genome of the virus VAL (8990-9409) (E2-M) and cDNA gene (ODC);

fragment size: 420 BP fragment of cDNA of the gene of the viral protein E2-M;

website cloning: ODC for sites NheI-SaI 1; cDNA fragment of the gene of the virus VAL on BstXI site, located between the two plots PEST gene equivalents ODC;

and intended for the development of DNA vaccines against the virus VAL.



 

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