Expression vector encoding coronavirus-like particle

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and virology. The expression vector for cloning Class I viral fusion protein gene contains Coronavirus M protein gene, Coronavirus E protein gene, IRES site, a first eukaryotic promoter linked to the M protein gene, Coronavirus Spike gene and MCS site and a second eukaryotic promoter linked to the Spike gene.

EFFECT: vector can be used as DNA vaccine candidate against virus infection diseases.

18 cl, 1 dwg, 4 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

This invention relates to the field of recombinant DNA technology, more specifically to the field of DNA vaccines. In particular, this invention relates to a new expressing vector codereuse virus-like particle, for cloning viral gene fused protein Class I and to use this vector as a candidate DNA vaccines.

The LEVEL of TECHNOLOGY

In the past 30 years, vaccination has played a key role in the control of viral diseases. Vaccination is based on the simple principle of immunity: as soon as the animal is exposed to an infectious agent, it sets the immune protection (immunity) against infection by the same agent. The goal of vaccination is to induce the animal to establish this defense to infection. This is usually performed through the use of live attenuated or killed form of a specific virus as immunogens. The success of these approaches in the past was due, in part, presentation of the native antigen and the ability of the attenuated virus to induce the full range of immune responses obtained in the case of this infection. However, the conventional vaccine methodology were always subject to potential limitations. Attenuated strains can mutate, becoming the more virulent or non-immunogenic; improperly inactivated vaccines can cause disease, they must prevent.

Recombinant DNA technology provides the ability to exclude some of these disadvantages of conventional vaccines, making possible the development of vaccines based on the use of specific antigens, but not the intact infectious agent, as immunogens. These methods include a peptide vaccine consisting of chemically synthesized, immunoreactive epitopes; subunit vaccines produced by expression of viral proteins in recombinant heterologous cells; and the use of live viral vectors for the presentation of one or more specific antigens. As peptide and subunit vaccines is subject to several potential limitations. The main problem is the difficulty of ensuring that the conformation of the constructed protein mimics the conformation of these antigens in their natural environment. For boosting this immune response shall be a suitable adjuvants and, in the case of peptides, carrier proteins. In addition, these vaccines induce primarily humoral response and, therefore, may not induce effective immunity.

Numerous other methods have been developed for introducing new genetic information into target cells. At the present time is I the most effective way of introducing DNA into target cells is the use of modified viruses the so-called recombinant viral vectors. The most commonly used system for viral vectors based on retroviruses, adenoviruses, herpes viruses, or adeno-associated viruses (AAV). All systems have inherent advantages and disadvantages. Some of these vector systems have the ability to integrate their DNA into the genome of the host cell, while others do not have this ability. In the case of some vector systems viral genes can be completely removed from the vector, whereas in other systems, it is not yet possible. A number of vector systems have very good properties delivery in vivo, while other systems do not have such properties. Some types of vectors is very easy to get in large quantities, while others are extremely difficult to obtain.

Coronaviruses are three or four protein in their membranes. Protein M is the most abundant component. Small protein E is a minor, but significant viral component. The importance of protein S in the pathogenesis is consistent with its biological function as the penetration and spread of the virus (Collins, A.R., et al, 1982, Virology 119:358-371; Williams, R.K., et al., 1991, Proc. Natl. Acad. Sci. USA 88:5533-5536). When the expression on the membrane of the virion protein S binds to the cellular receptor and induces the fusion of viral and cellular membranes during p is unknowne virus. After infection protein S, expressed on the plasma membrane of infected cells, induces the fusion of cells. Protein S also plays a role in the immune response to viral infection as a target for neutralizing antibodies (Collins, A.R., et al., 1982, Virology 119:358-371) and as an inducer of cell-mediated immunity (Bergmann, S., et al., 1996, J. Gen. Virol. 77:315-325; Castro, R.F., and S. Perlman, 1995, J. Virol. 69:8127-8131). Proteins M and E are the minimum protein units for virus Assembly (Baudoux, P., et al., 51998, J. Virol. 72:8636-8643; Bos, E.G., 1996, Virology 218:52-60; de Haan, C.A.M, et al., 1998, J. Virol. 72:6838-6850; Godeke, G.-J, et al., 2000, J. Virol. 74:1565-1571; Vennema H., et al., 1996, EMBO J. 15:2020-2028). Both of these protein are integral membrane proteins. Simultaneous expression of proteins M and E is sufficient to start the formation of virus-like particles (the VLP). While expression of protein S protein M and E. this protein E is included in the VLP with the supposedly authentic conformation. It was now demonstrated for hepatitis virus of mice (MHV) (Bos, E.G., 1996, Virology 218:52-60; de Haan, CAM., et al, 1998, J. Virol. 72:6838-6850; Vennema H, et al, 1996. EMBO J. 15:2020-2028)virus, transmissible gastroenteritis (Baudoux, P., et al., 51998, J. Virol. 72:8636-8643) virus and feline infectious peritonitis (Godeke, G.-J., et al., 2000, J. Virol. 74:1565-1571). There is a hypothesis that the coronavirus membrane consists mainly of dense matrix lateral interacting Belgium, that in some way requires protein for active viral replication as a result of penetration into the cell (bazinga) and in which the glycoproteins's NOT, if they are available, specific interactions of M through the carboxy end and the transmembrane region Spike (de Haan, et al, 1999, J. Virol. 73:7441-7452; Ngiyen, V-P, and B.G. It, 1997. J. Virol. 71:9278-9284; Opstelten, D.-J.E, et al., 1995, J. Cell Biol. 131:339-349; Vennema H., et al., 1996, EMBO J. 15:2020-2028) Such Spike-containing the VLP can infect cells with infectious, similar infectivity of authentic virus (Bos, E.G., 1996, Virology 218:52-60). However, not developed effective DNA vectors for use as DNA vaccines.

Thus, there is a need for the development of efficient DNA vector encoding virus-like particles, which can provide functional viral protein in the surface of the VLP, and the VLP these will be an excellent candidate as a potential vaccine against viral infectious diseases.

The INVENTION

This invention relates to expressing vector for cloning viral gene fused protein Class I and apply as a candidate DNA vaccine expressing this vector contains:

i) a first transcription unit containing a gene of a protein membrane (gene protein M) Coronavirus, envelope protein gene (gene protein (E) Coronavirus and inside the affected state sequence of the site of entry of ribosomes (IRES), where IRES is built into the junction gene protein-membrane and envelope protein gene;

ii) the first eukaryotic promoter functionally linked to the gene of the protein of the membrane, where the first promoter is located to the left (against the course of transcription from protein gene M and triggers the expression of this first transcription units;

iii) a second transcription unit containing a gene SpikeCT Coronavirus and a multiple cloning site (MCS) for cloning or embedding in the reading frame of the gene viral fused protein Class I, where the MCS is located at the beginning of the gene SpikeCT and has the cutting sites of restriction enzymes; and

iv) a second eukaryotic promoter functionally linked to a gene SpikeCT, where this second promoter is located to the left (against the course of transcription from a gene SpikeCT and triggers the expression of this second transcription units;

where the transcriptional activity of the first eukaryotic promoter is stronger than the transcriptional activity of the second eukaryotic promoter.

BRIEF DESCRIPTION of DRAWING

The drawing shows a plasmid map of one preferred variant implementation of expressing the vector of this invention.

DETAILED description of the INVENTION

Genes encoding viral polypeptides capable of self-Assembly in defective, not self-replicating viral cha is based, can be obtained from genomic DNA DNA virus or genomic cDNA RNA-virus or available subgenomic clones containing these genes. These genes include the genes encoding the viral capsid proteins (i.e. proteins that form the viral protein shell), and, in the case of having the shell of viruses, such as retroviruses, genes encoding the viral envelope glycoproteins. These virus-like particles can be selected and used themselves as immunogens for vaccination against pathogenic viruses or for therapeutic purposes, such as enhancing immune responses in the infected individual, or for targeted delivery of therapeutic agents such as cytotoxic drugs to specific cell types.

This invention provides expressing vector for cloning viral gene fused protein Class I and apply as a candidate DNA vaccine expressing this vector contains:

i) a first transcription unit containing a gene of a protein membrane (gene protein M) Coronavirus, envelope protein gene (gene protein (E) Coronavirus and internal consistency of the site of entry of ribosomes (IRES), where IRES is built into the junction gene protein-membrane and envelope protein gene;

ii) the first eukaryotic promoter functionally linked to the gene of the protein of the membrane is, where the first promoter is located to the left (against the course of transcription from protein gene M and triggers the expression of this first transcription units;

iii) a second transcription unit containing a gene SpikeCT Coronavirus and a multiple cloning site (MCS) for cloning or embedding in the reading frame of the gene viral fused protein Class I, where the MCS is located at the beginning of the gene SpikeCT and has the cutting sites of restriction enzymes; and

iv) a second eukaryotic promoter functionally linked to a gene SpikeCT, where this second promoter is located to the left (against the course of transcription from a gene SpikeCT and triggers the expression of this second transcription units;

where the transcriptional activity of the first eukaryotic promoter is stronger than the transcriptional activity of the second eukaryotic promoter.

According to this invention, expressing this vector contains a first transcription unit containing a gene of a protein membrane (gene protein M) Coronavirus, envelope protein gene (gene protein (E) Coronavirus and internal consistency of the site of entry of ribosomes (IRES), where IRES is embedded in the connection of the gene of the protein of the membrane and the gene of the protein shell.

According to this invention, the gene of the protein shell of this invention encodes the envelope protein (protein E) Coronavirus. This protein E is small, associated the data with the membrane protein. Protein S is a minor, but significant viral component. In the cells it accumulates in the membranes and induces adhesion of membranes intermediate compartment (IC), leading to the emergence of typical structures. Some of these proteins appears in extracellular membrane structures of unknown identity. Preferably, the cov of this invention is a coronavirus pigs, humans and birds. More preferably, this coronavirus is a coronavirus TGEV pigs, coronavirus A person or SARS virus (severe acute respiratory syndrome) person. More preferably, the gene of the protein E of this invention has the sequence presented in SEQ ID NO:1.

According to this invention, the gene of the protein of the membrane of this invention encodes a protein membrane protein M) Coronavirus. This protein M is the most abundant component; it is a glycoprotein of type III, consisting of short aminoanisole of ectodomain, three consecutive transmembrane domains and a long carboxykinase domain inside the virion (or in the cytoplasm). Preferably, the cov of this invention is a coronavirus pigs, humans and birds. More preferably, this coronavirus is a coronavirus TGEV pigs, coronavirus A person or SARS virus (severe OS is cerned respiratory syndrome) person. More preferably, the gene of the protein E of this invention has the sequence presented in SEQ ID NO:2.

According to this invention for assembling shell of coronavirus is only necessary protein M protein E (Cornelis A.M. de Haan et al., Journal of Virology, June 2000, p.4967-4978). Expression in cells of genes encoding these proteins leads to the formation and release of virus-like particles (the VLP), similar in size and shape with authentic virions.

According to this invention, the first transcription unit contains an internal sequence of the site of entry of ribosomes (IRES), which is built into the junction gene M gene E. IRES makes it possible to broadcast two or more proteins from two - or polycistronic mRNA. Unit IRES merge with the 5'-ends of one or more coding sequences, which are then inserted into these vectors at the end of the initial coding sequence, so that the coding sequence is separated from other sequences by the IRES. According to this invention, any derived IRES can also be used in plasmid constructions of this invention. The IRES sequence allows the apparatus ribosomes to initiate translation from a secondary site in a single transcript.

According to this invention Express yuushi vector of this invention contains the first eukaryotic promoter, functionally linked to the gene of the protein of the membrane, where the first promoter is against the course of transcription (left) from gene protein M and starts the first expression of the transcription unit. Preferably, the first eukaryotic promoter is derived from a virus promoter, such as CMV, SV40, RSV, LTR of HIV-1 and hybrid enhancer promoter, beta-actin/CMV promoter and promoter specific for muscle desmin, creatine kinase, promoter, beta-actin and other ubiquitous promoter expression, such as promoter EF1-alpha promoter of ubiquitin, Most preferably, the first eukaryotic promoter is pCMV, hybrid enhancer promoter, beta-actin/CMV promoter, the promoter of beta-actin.

According to this invention, the second transcription unit contains a gene SpikeCT and a multiple cloning site (MCS)with the cutting sites of restriction enzymes, where the MCS website localized at the beginning of the gene SpikeCT. Gene SpikeCT interacts with domain interacting protein M, which encodes a C-terminal heptanal (7-fold) repeat located to the left of the rich aromatic residue region adjacent to the transmembrane segment of the Spike protein of the coronavirus. A multiple cloning site (MCS) has multiple cutting sites of restriction enzymes. Site cutting restriction enzymes on the includes, but not limited to, SmaI, BsaBI, EcoRV and BspEI. MCS is located at the beginning of the gene SpikeCT and can be used for cloning or embedding in the reading frame of the gene viral fused protein Class I viral Gene fused protein Class I includes, but is not limited to, Dr HIV, influenza virus, and the Spike of the SARS virus. Gene viral fused protein Class I derived from viruses that adapt viral mechanism merger of Class I and include coronavirus, HIV and influenza virus, the gene is introduced into clone/expressing the vector of this invention, and then this vector is used as candidate DNA vaccines. Preferably, the gene SpikeCT has the sequence presented in SEQ ID NO:3.

The CODING sequence of the GENE SpikeCT (SEQ ID NO:3)

According to this invention, expressing the vector of this invention can produce virus-like particle and Express functional fused gene of the virus Class I on the surface of virus-like particles. Thus, multiple cutting sites of restriction enzymes (multiple cloning site (MCS), including, but not limited to, SmaI, BsaBI, EcoRV and BspEI, located at the beginning of the gene "SpikeCT" and these sites can be used to embed or cloning viral gene fused protein Class I clone/expressing in ctor of this invention and then this vector can be used as candidate DNA vaccines. Viruses that adapt viral mechanism for merging Class I, include, but are not limited to, corona virus, HIV, influenza virus, etc.

According to this invention, the second promoter is against the course of transcription (left) from gene SpikeCT and triggers the expression of the second transcription unit. Preferably, this second eukaryotic promoter is derived from a virus promoter, such as CMV, SV40, RSV, LTR of HIV-1 and hybrid enhancer promoter, beta-actin/CMV promoter and promoter specific for muscle desmin, creatine kinase, promoter, beta-actin and other ubiquitous or constitutive promoter expression, such as promoter EF1-alpha promoter of ubiquitin. Most preferably, the second eukaryotic promoter is pCMV, SV40, promoter, beta-actin and promoter EF1-alpha.

According to this invention, a transcription unit of this invention include the poly-a signal. Persons with qualifications in this field it is clear that any transcription has the poly-a signal. Preferably, the transcriptional unit of this invention include the poly-a signal BGH.

According to this invention, expressing this vector contains the site of initiation of replication for propagation of the plasmid in the cell host. The definition of the site of initiation of replication depends on the types of host cells.

Under this is obreteniyu expressing the vector of this invention further comprises a gene of resistance to antibiotics as a breeding marker.

According to this invention, expressing the vector of this invention can spontaneously to form virus-like particles. These virus-like particles can be used as candidate vaccines against viral infections. These virus-like particles do not contain genetic material of the virus. Thus, such particles are not infectious ability. Expressing the vector of this invention can be administered to animals and transcribed by the cell-hosts for the production of virus-like particles.

Most viruses consist of a small number of proteins only with the structural limitations imposed on them. In fact, using this small number of proteins viruses have to generate almost crystalline, highly repetitive surface. Such highly repetitive antigens effectively sew receptors of b-cells in a process that generates a strong signal activation in b cells. In contrast, the autoantigens are usually not highly organized, in particular, the autoantigens that are available In cells. Thus, b cells use the organization of antigens as a marker for infectious non-autoantigen. Vaccines based on virus-like particles (the VLP) use this basic phenomenon, because the VLP detect surface is STI, the same repetitive and organized, and that the surface of the virus. Consequently, like a virus, the VLP is capable of running a strong b-cell response in the absence of adjuvants. Based on the above principle, virus-like particles produced by expression expressing the vector of this invention in cells, can be used as candidate vaccines against viral diseases.

EXAMPLES

Gene E, synthesized using PCR

For the synthesis of gene E of the SARS virus (severe acute respiratory syndrome) used polymerase chain reaction. Matrix PCR (0.1 pmol) gene E of the SARS virus is a mixture of primers, listed below, and the PCR reaction was performed using standard PCR method described by Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press), using Taq polymerase KOD (Novagene.com). The PCR primers described below:

DNA template was first denaturiruet at 95°C for 3 minutes. The PCR conditions are listed below as follows:

The first PCR reaction with 10 cycles of 3 steps:

1. Annealing: 58°C for 20 seconds.

2. Elongation: 72°C for 40 seconds.

3. Denaturation: 95°C for 1 minute.

The second PCR reaction with 20 cycles of 3 steps:

1. Denaturation: 95°C for 1 minute.

2. Annealing: 62°C for 20 seconds.

3. Untinen is: 72°C for 40 seconds.

The obtained double-stranded full-size gene products were analyzed by 1.2% agarose gel and purified using the cleaning kit QIAquick PCR (Qiagen Inc.) and then ligated with the vector pGEM-T (Promega Co.) to obtain a clone pGEM(T)/EA+. The sequence of a gene E. presented in SEQ ID NO:1.

Gene M, synthesized using Spellsinger-PCR

Synthesis gene M of the SARS virus (severe acute respiratory syndrome) was similar to the PCR method described above, the matrix PCR (0.1 pmol) gene E of the SARS virus is a mixture of primers, listed below, and the PCR reaction was performed using Taq polymerase KOD (Novagene.com as follows:

Used the following PCR primers:

The PCR conditions were essentially the same as above. The obtained double-stranded full-sized products of the gene M were analyzed by 1.0% agarose gel and purified using the kit for purification or extraction from the gel (QIAquick PCR (Qiagen Inc.) and then ligated with the vector pGEM-T (Promega Co.) to obtain a clone pGEM(T)/M. the Sequence of the gene M is presented in SEQ ID NO:2.

Gene "SpikeCT"encoding heptanal district 2 and the transmembrane domain protein gene of coronavirus Spike was synthesized using PCR.

The method and conditions of PCR, purification, and CL is the treatment of PCR products are the same as explained above. Matrix PCR (0.1 pmol) gene SpikeCT is a mixture of primers, listed below.

Used the PCR primers listed below:

Used PCR conditions listed above. The obtained double-stranded full-sized products of the gene SCT320 was purified using the cleaning kit Qiagen/PCR (Qiagen Inc.) and cloned using the vector pGEM(T) (Promega Co.) to obtain pGEM(T)/SpikeCT (EcoRV/BstBI). Plasmid pGEM(T)/SpikeCT (EcoRV/BstBI) contains a gene that encodes the C-terminal heptanal repeat located to the left of the rich aromatic residue region adjacent to the transmembrane segment of the Spike protein of the coronavirus. The coding sequence of the gene "SpikeCT represented in SEQ ID NO:3.

The construct expressing the vector of this invention

Manipulation of DNA described in Sambrook and Russel et al., Molecular cloning, third edition, Cold Spring Harbor Laboratory Express. Restrictase, DNA T4 ligase, the enzyme maple bought from New England Biolab.com and used in accordance with manufacturer's recommendation.

I. Construction of plasmid gene M of the SARS virus, run by the promoter pCMV

Clone pGEM(T)/M was used as DNA template for PCR with primers T7 promoter and SP6, and PCR conditions, except that the annealing temperature was set at 50°C for 20 seconds, are what akimi, what is described above. The obtained PCR products of the gene M was purified using the cleaning kit Qiagen/PCR (Qiagen Inc.) and uncoupled BcII and NotI to obtain DNA inserts containing gene M. SARS. Plasmid pEGFP-N1 (Clontech Co.) split BgIII and NotI and the resulting split BgIII, NotI plasmid pEGFP-N1 was used as a DNA vector for ligation with split BcII, NotI DNA inserts of a gene M. SARS with obtaining DNA plasmids pN1/M. Plasmid pN1/M were digested NheI and NotI to obtain DNA inserts containing gene M. SARS. Plasmid Rast (Promega Co.) were digested NheI and NotI. The obtained split NheI, NotI DNA vector Rast ligated with split NheI, NotI DNA inserts of a gene M. SARS with obtaining DNA plasmids Rast/M DNA plasmid Rast/M BgIII digested and Asp718 with obtaining DNA inserts containing the CMV promoter and gene M. SARS. Plasmid pCDNA3.1(+) (Invitrogen Co.) split BgIII and Asp718. The obtained split BgIII, Asp718 DNA vector pCDNA3.1(+) ligated with split BgIII, Asp718 DNA containing inserts of the CMV promoter and gene M. SARS, obtaining DNA plasmid PCDNA3.1+M.

II. Construction of plasmid gene E. IRES-SARS

Plasmid plRES2-EGFP (Clontech Co.) split XhoI and NcoI to obtain DNA inserts "IRES". Vector GL3basic (Promega Co.) split XhoI and NcoI. Split XhoI, NcoI DNA vector GL3basic ligated with split XhoI, NcoI DNA insert IRES using DNA T4 ligase (NEB Co.). Mixture for ligation was transformed into competent cells of E. coli H5 obtaining DNA plasmids pGL3B/IRES-luciferase. Clone T/EA+containing gene E. SARS with insertional mutation of the second alanine, were digested NcoI and XbaI to obtain DNA inserts of a gene E. SARS. DNA plasmids pGL3B/IRES-luciferase were digested NcoI and XbaI. This split NcoI, XbaI DNA pGL3B vector/IRES-luciferase ligated with split NcoI, XbaI DNA inserts of a gene EA+SARS obtaining DNA plasmids pGL3B/IRES-EA+.

III. Construction started expressing pCMV plasmid genes M+E (expressing vector Coronavirus-like particles (CoVLP))

DNA plasmids pGL3B/IRES-EA+were digested BamHI and XbaI to obtain DNA inserts containing the IRES gene EA+SARS. DNA plasmid pCDNA3.1+M was digested BamHI and XbaI. The obtained split BamHI, XbaI DNA vector pCDNA3.1+M ligated with split BamHI, XbaI DNA inserts containing the fused gene is IRES and EA+SARS, obtaining the plasmid pCDNA3.1+M/IRES-EA+.

IV. Construction of DNA-vectors of this invention

Plasmid pGEM(T)/SpikeCT (EcoRV/BstBI), which contains the gene that encodes the C-terminal heptanal repeat located to the left of the rich aromatic residue region adjacent to the transmembrane segment of the Spike protein of the coronavirus, were digested Spel and built using enzyme maple and dNTP. After separation on a 1.2% agarose gel, this DNA fragment was purified using a kit for the extraction of the gel with QIAquick (Qiagen Inc.) and additionally BstBI digested. Obtained the fragments of the gene "SpikeCT" was used as the DNA inserts and ligated with split BsaBI, BstBI, purified from the gel with a DNA vector plasmid pCDNA3.1+M/IRES-EA+. After transformation into cells of E. coli DH5 received a DNA vector of the invention, the so-called CoVLP-cloning vector. There are sites cutting several restriction enzymes (multiple cloning site (MCS), including SmaI, BsaBI, EcoRV, BspEI, located at the beginning of the gene "SpikeCT", and these sites can be used to clone fused in reading frame viral gene fused protein Class I gene SpikeCT in the vector of the invention and then the DNA plasmid can be used as candidate DNA vaccines. Map CoVLP-cloning vector shown in the drawing. The sequence CoVLP-cloning vector represented in SEQ ID NO:49.

The DNA sequence of the cloning vector CoVLP

(pCDNA3.1+M+IRES+SpikeCT) (SEQ ID NO:49))

1. Expressing the vector for cloning viral gene fused protein Class I and apply as a candidate DNA vaccine expressing this vector contains:
i) a first transcription unit containing a gene of a protein membrane (gene protein M) Coronavirus, envelope protein gene (gene protein (E) Coronavirus and internal consistency of the site of entry of ribosomes (IRES), where IRES is embedded in months what about the connection of the gene of the protein of the membrane and envelope protein gene;
ii) the first eukaryotic promoter functionally linked to the gene of the protein of the membrane, where the first promoter is located to the left (against the course of transcription from protein gene M and triggers the expression of this first transcription unit;
iii) a second transcription unit containing a gene SpikeCT Coronavirus and a multiple cloning site (MCS) for cloning or embedding in the reading frame of the gene viral fused protein Class I, where the MCS is located at the beginning of the gene SpikeCT and has the cutting sites of restriction enzymes; and
iv) a second eukaryotic promoter functionally linked to a gene SpikeCT, where this second promoter is located to the left (against the course of transcription from a gene SpikeCT and triggers the expression of this second transcription units;
where the transcriptional activity of the first eukaryotic promoter is stronger than the transcriptional activity of the second eukaryotic promoter.

2. Expressing the vector according to claim 1, where this Coronavirus is a coronavirus pigs, humans and birds.

3. Expressing the vector according to claim 1, where this Coronavirus is a coronavirus TGEV pigs, coronavirus A person or SARS coronavirus person.

4. Expressing the vector according to claim 1, where the gene envelope protein has the sequence presented in SEQ ID NO:1.

5. Expressing the vector according to claim 1, where GE is membrane protein has the sequence presented in SEQ ID NO:2.

6. Expressing the vector according to claim 1, where the gene SpikeCT has the sequence presented in SEQ ID NO:3.

7. Expressing the vector according to claim 1, which further comprises the site of initiation of replication for propagation of the plasmid in the cell host.

8. Expressing the vector according to claim 1, which additionally contains a gene for resistance to the antibiotic as breeding token.

9. Expressing the vector according to claim 1, where the first and second transcription units have the poly-a signal.

10. Expressing the vector of claim 9, where the poly-a signal is the poly-a signal BGH.

11. Expressing the vector according to claim 1, which has the sequence presented in SEQ ID NO:49.

12. Expressing the vector according to claim 1, where the gene SpikeCT encodes the C-terminal heptanal repeat located to the left of the rich aromatic residue region adjacent to the transmembrane segment of the Spike protein of the coronavirus.

13. Expressing the vector according to claim 1, where the multiple cloning site contains the restriction site selected from the group consisting of Smal, BsaBI, EcoRV and BspEI.

14. Expressing the vector according to claim 1, where viral gene fused protein Class I is selected from the group consisting of gp160 HIV, influenza virus and Spike of SARS.

15. Expressing the vector according to claim 1, where the first eukaryotic promoter selected from the group consisting of CMV promoters, SV4, RSV, LTR of HIV-1, hybrid enhancer promoter, beta-actin/CMV promoter, promoter-specific muscles desmin, creatine kinase, promoter of the beta-actin promoter, EF1-alpha promoter of ubiquitin.

16. Expressing the vector according to claim 1, where the first eukaryotic promoter selected from the group consisting of CMV promoter, a hybrid enhancer promoter, beta-actin/CMV promoter and promoter beta-actin.

17. Expressing the vector according to claim 1, where the second eukaryotic promoter selected from the group consisting of promoters CMV, SV40, RSV, LTR of HIV-1, hybrid enhancer promoter, beta-actin/CMV promoter, promoter-specific muscles desmin, creatine kinase, promoter of the beta-actin promoter, EF1-alpha promoter of ubiquitin.

18. Expressing the vector according to claim 1, where the second eukaryotic promoter selected from the group consisting of pCMV promoter, SV40, beta-actin and EF1-alpha.



 

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FIELD: biotechnology, virology, medicine.

SUBSTANCE: invention relates to attenuated virus derived from modified Ankara vaccina virus. Said virus are not able for reproduction by replication in human cell lines. Also disclosed are application of virus or recombinant variants thereof as drug or vaccine, as well as method for inducing of immune response in patients with defected immunity, in patients having immunity to vaccine virus, or in patient during antiviral therapy.

EFFECT: variant of Ankara vaccina virus effective in medicine and veterinary.

86 cl, 15 dwg, 1 tbl, 2 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, virology, medicine.

SUBSTANCE: invention relates to attenuated virus derived from modified Ankara vaccina virus. Said virus are not able for reproduction by replication in human cell lines. Also disclosed are application of virus or recombinant variants thereof as drug or vaccine, as well as method for inducing of immune response in patients with defected immunity, in patients having immunity to vaccine virus, or in patient during antiviral therapy.

EFFECT: variant of Ankara vaccina virus effective in medicine and veterinary.

86 cl, 15 dwg, 1 tbl, 2 ex

FIELD: gene engineering.

SUBSTANCE: the present innovation deals with the ways for obtaining transgenic poultry due to introducing retroviral vectors into blastodermal cells through the fissure in the shell of nonhatching egg from the side of its blunt end. With the help of insulin syringe one should introduce gene constructions for the depth of about 2-3 cm near a germinal disk. The innovation enables to simplify the procedure of introducing gene constructions into target cells at maintaining general efficiency of transgenesis that leads to the decrease of embryonic lethality.

EFFECT: higher efficiency.

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