Agent for neutralising smallpox virus

FIELD: medicine.

SUBSTANCE: invention represents an agent for neutralising smallpox virus representing an artificial single-chain human 1A antibody having an amino acid sequence presented in the claim material, and exposed on a surface of the filamentous phage M13.

EFFECT: invention enables neutralising smallpox virus.

7 dwg, 4 ex

 

The invention relates to a tool to neutralize variola virus on the basis of single-stranded human antibodies constructed in vitro on the basis of the genes encoding the variable domains of immunoglobulins people vaccinated with the vaccinia virus, recombinant hamidou DNA pHEN-1A, containing the result of genetic engineering methods artificial gene single-stranded antibodies person under the control of the Lac promoter of the operon, providing in Escherichia coli cells the synthesis of single-stranded human antibodies in the composition of the chimeric protein envelope protein p3 of the bacteriophage M13, capable of neutralizing the orthopoxviruses, and single-chain antibody human 1A exposed on the surface of filamentous bacteriophage M13 in the composition of the envelope protein p3, capable to neutralize variola virus, vaccinia virus and variola virus cows, and can be used in medicine, biotechnology, protein and genetic engineering.

The genus is classified in the genus orthopoxvirus, which is from the poxviridae family, involves complex DNA-containing viruses that can replicate in the cytoplasm of cells of vertebrates and invertebrates. To this genus are pathogenic human viruses variola virus and Monkeypox virus, viruses, normally only cause local infection, vaccinia virus and variola virus cows, and non-pathogenic to man the ESA viruses virus ectromelia, the smallpox virus camels and others [1]. It should be noted that in people with weakened immune status viruses ospowiki and smallpox cows can cause severe illness or even generalized infection.

In connection with the termination of the natural transmission of the virus of smallpox was discontinued mass vaccination with vaccinia virus. However, despite the eradication of smallpox, there are reasons why the orthopoxviruses continue to be a source of biological hazards to people, and the natural or deliberate spread of smallpox may contribute to the fact that currently the majority of the population has no immunity to these viruses. In addition, the resumption of mass vaccination with vaccinia virus in the United States confirmed the inevitability of the emergence of post-vaccination complications in some cases, especially that the majority of adults have not been vaccinated vaccinated spooktinu in childhood [2].

Vaccination with vaccinia virus, giving reliable protection [3], can be accompanied by complications, especially in people with congenital or acquired immunodeficiency, there is therefore a need to develop specific means of prevention of post-vaccination complications. Currently, this tool is whey vaccine IMM is noglobulin (VIG) [1], however, the use of drugs derived from human blood, is always accompanied by certain biological risk, besides this medication roads and inaccessible. Alternative vaccine immunoglobulin could be recombinant human antibodies specific to orthopoxviruses.

Currently, the most preferred are fully human recombinant antibody (fully human antibodies). To create them using the merge variable domains of human antibodies with the target activity, with constant domains of human immunoglobulins of the desired isotype. One way to obtain a variable domains is their selection from a combinatorial phage libraries mini-antibodies person. It is important that the selected variable domains possessed neutralizing properties against the target virus. To date, such a method is designed with full-length human antibodies against a number of viral agents[4, 5, 6, 7]. Known designed in such a way that a full-sized human antibodies against orthopoxviruses [8, 9, 10]. These antibodies were able to bind viruses ospowiki smallpox cows [8, 9, 10] and neutralize vaccinia virus [10]. Antibodies b9 and C4 are also able to inhibit the infectivity of Monkeypox virus [10]. Full-length human antibodies with neutralizing and protective properties against vaccinia virus, were obtained using transgenic mice [11].

The closest analogue (prototype) is the antibody A [12], selected for ability to bind the smallpox virus cows and to inhibit the infectivity of viruses ospowiki and smallpox of the cow's immune combinatorial fahmideh library of single-chain antibodies man, constructed in vitro on the basis of the genes encoding the variable domains of immunoglobulins people vaccinated with vaccinia virus [13].

However, in the above technical solutions [8-12] properties to inhibit the infectivity of the virus of smallpox for antibodies A, b9 and C4 was not shown.

The technical result of the claimed invention is to provide use of phage display plasmid DNA pHEN-1A for synthesis in cells of Escherichia coli single-stranded human antibodies 1A exposed on the surface of bacteriophage MC in the composition of the envelope protein P3, and is capable of neutralizing the infectivity of variola virus, vaccinia virus and variola virus cows.

This technical result is achieved by selection of fahmideh collection of recombinant fahmideh DNA pHEN-1A containing a unique gene single-stranded human antibodies 1A, can neutralize variola virus, vaccinia virus and variola virus cows; obtaining Advocaat knogo human antibodies 1A, exposed on the surface of bacteriophage MC in the composition of the envelope protein P3, and is capable of neutralizing the infectivity of variola virus, vaccinia virus and variola virus cows.

The invention consists in the following:

- from the immune ragovoy library selected fahmida DNA pHEN-1A containing a unique gene single-stranded human antibodies 1A, can neutralize variola virus, vaccinia virus and variola virus cows;

- selected single-stranded phage antibody 1A exposed on the surface of filamentous bacteriophage MC in the composition of the envelope protein P3, capable of neutralizing the virus of smallpox, ospowiki and smallpox of the cow.

Methods of phage display selected single-chain antibody human 1A exposed on the surface of filamentous bacteriophage MC in the composition of the envelope protein P3 encoded fahmideh DNA pHEN-1A. The resulting antibody is able to neutralize variola, ospowiki and smallpox of the cow.

To generate antibodies against orthopoxviruses, such as virus ectromelia, spend affinity enrichment combinatorial immune library specific clones. A combinatorial library of antibodies is a collection of bacteriophages, each of which exhibits on its surface a unique od is acapodene antibody in the composition of the shell phage protein [14]. The enrichment of the libraries using virus ectromelia as antigen with subsequent selection of individual clones capable of producing bacteriophages, exposing on the surface of single-chain antibodies against the virus of ectromelia. For this purpose, an aliquot of the transformed Escherichia coli cells is thawed, to raise srednetehnicheskoy phase and infecting phage helper MC, you get a population of filamentous bacteriophage, each of which exhibits on its surface a unique single-chain antibody man in the form of a chimeric protein with envelope protein p3 phage MK.

Affinity enrichment of the library is carried out in two consecutive rounds. For the first round of enrichment in the wells of polystyrene tablet adsorb virus ectromelia (strain K-1) at a concentration of 100 μg/ml At the end of the sorption designated saturate nonspecific binding with 5%solution of skimmed milk powder in phosphate-buffered saline (PBS), after which each well add an aliquot (1011The COMBAT) library as a collection of filamentous bacteriophages. Nonspecific bound peroxidase phage particles can be removed by washing PBS, and phages bearing on their surface a single-chain antibodies, capable of contacting the virus ectromelia, elute solution to tritium is on. The obtained eluate infect cell culture E. coli TG1 in a stage of exponential growth for amplification buervenich phage antibodies.

The second round of affinity enrichment carried out similarly. The concentrations of virus ectromelia reduced to 50 µg/ml, respectively. The degree of enrichment of the library with antibodies specific to the virus ectromelia, check using indirect enzyme-linked immunosorbent assay (ELISA) for the ability of populations of phage antibodies obtained from each round of affinity enrichment, to bind the virus ectromelia (figure 1). When this phage antibodies distinguish, as described in Example 1. As a negative control, use linking populations of phage antibodies adsorbed 5% skim milk in PBS.

Next, from the population of phage antibodies enriched against virus ectromelia, conduct the selection of monoclonal phage antibodies specifically binding the virus ectromelia, using ELISA. Virus ectromelia adsorb on 96-well immunological tablets (metroliner", Russia) at a concentration of 3 μg/ml Designated nonspecific binding blocked with a solution of 3%bovine serum albumin (BSA, Sigma) in phosphate buffer, pH 7.2. Then in the wells contribute allocated as described in Example 1, phage antibodies, diluted with PBS containing 0.1% tween, and incubi the comfort of 1 hour at 37°C. After washing the wells contribute polyclonal anti-M13 antibody rabbit in a dilution of 1:16000, and then individuai conjugate alkaline phosphatase (Sigma) at a dilution of 1:4000. As a Chromogen use paranitrophenylphosphate. Control of nonspecific binding is the binding of the virus by phage helper MC, not bearing fragments of antibodies on their surface. As a negative control using binding phage antibodies with BSA. Clones showing a signal whose value is 3 times or more than the control of nonspecific binding and the negative control, is taken as positive (figure 2).

Selected phage antibodies isolated and examined using ELISA in serial dilution of antibodies. At the same time as antigens for a more complete characteristics of the antibodies can be used in addition to virus ectromelia other orthopoxviruses, such as viruses ospowiki and smallpox of the cow (figure 3).

Standard analysis of the neutralizing activity carried out for all of phage antibodies specifically binding the virus ectromelia. This test verifies the ability to inhibit plaque formation by the variola virus, ospowiki and smallpox cows on the monolayer of cells Vero E6. As a negative control using bacteriophage-assistant MC, no bearing on what VOA the surface of single-chain antibodies. The ability to neutralize viral infectivity appreciate the neutralization titer, defined as the dilution of phage antibodies, demonstrating the decrease in the number of viral plaques by not less than 50% (figure 4).

Next, perform a determination of the nucleotide sequences of the genes encoding single-chain antibody, neutralizing viral infectivity. For amplification of the gene encoding single-chain antibody 1A, as a matrix, use famiglie DNA extracted by the method of alkaline lysis [15], and oligonucleotide primers: LMB 5'-CAGGAAACAGTCATGAC, pHEN-SEQ 5'-CTATGGGGCCCCATTCA. Amplification is performed by PCR using TaqSE DNA polymerase at a temperature of annealing of primers LMB and pHEN-SEQ 52°C. determination of the nucleotide sequences of the purified Vh and Vl fragments is carried out in both directions using an automatic sequencing machine (ABI 3730XL Genetic Analyser (Applied Biosystems) and set BigDye®Terminator v3.1 Cycling Sequencing Kit (Applied Biosystems, USA. For sequence analysis using the program Lasergene SeqMan, Vector NTI Suite 8 and MEGA 4.1. The obtained sequences are compared to sequences contained in the database NCBI BLAST. Figure 5 presents the nucleotide sequence and encoded by its amino acid sequence unique gene single-stranded human antibodies with virusray religouse activity against variola virus, ospowiki and smallpox of the cow, and figure 6 presents the amino acid sequence of this recombinant antibodies 1A.

Obtained from the described fahmideh collection of recombinant fahmida DNA pHEN-1A containing a unique gene single-stranded human antibodies 1A, capable of neutralizing the virus of smallpox, ospowiki and smallpox cows, characterized by the following features (Fig.7):

- has a molecular weight 3,47 MDA and size 5255 P.O.;

- encodes a single-chain antibody human 1A as part of a chimeric protein with envelope protein P3 phage MK;

- consists of the following elements:

a) DNA fragment, the size of 840 BP, including artificial gene single-stranded human antibodies 1A, capable of neutralizing the orthopoxviruses, in which the variable domain of the heavy chain is connected with the variable domain of the light chain of human immunoglobulin using DNA sequence encoding a flexible peptide linker Ser(Gly4Ser)2AlaArgGlySerGly4Ser, the sequence encoding the peptide is UNKNOWN, and having the nucleotide sequence of SEQ ID NO:1, are presented in figure 4;

b) fahmideh vector pHEN2 (MRC, UK), providing for efficient transcription of the obtained gene encoding single-chain antibody human 1A, and its expression;

contains:

a) the site of initiation of bulk packing : spindle, the purpose of ColE1 plasmid pBR322;

b) the promoter of the Lac-operon of E. coli;

C) genetic marker: AMPr gene of β-lactamase (bla), which determines the stability of the transformed fahmida pHEN-1A cells of Escherichia coli to ampicillin;

d) artificial gene single-stranded human antibodies 1A, including the sequence encoding the peptide is UNKNOWN, capable of neutralizing the orthopoxviruses, in which the variable domain of the heavy chain is connected with the variable domain of the light chain of human immunoglobulin using DNA sequence encoding a flexible peptide linker Ser(Gly4Ser)2AlaArgGlySerGly4Ser,

d) a unique recognition sites of the restriction endonucleases, with the following coordinates: HindIII(235), SfiI(328), NotI(1088), BamHI(1749), ClaI(2057), AvaI(2951), EcoRI(2379).

Thus, for the first time obtained plasmid DNA pHEN-1A for synthesis in cells of Escherichia coli single-stranded human antibodies 1A exposed on the surface of bacteriophage MC in the composition of the envelope protein P3, and is capable of neutralizing the infectivity of variola virus, vaccinia virus and variola virus cows.

The invention is illustrated by the following graphic materials presented on figure 1-7:

Figure 1. Linking the original library and populations of phage antibodies obtained after the 1st and 2nd rounds of affinity enrichment, with virus ectromelia (dark bars) is a 5% solution of skim milk in PBS (light bars) in ELISA.

Figure 2. Binding of phage antibodies with virus ectromelia (dark bars) and a 3% solution of BSA in PBS (light bars) in ELISA.

Figure 3. Binding of single-stranded phage antibodies 1A with various orthopoxviruses with serial dilutions of antibodies. Legend: 1 - the curve of binding antibodies 1A with virus ectromelia, 2 - binding antibodies 1A with vaccinia virus, 3 - binding antibodies 1A with smallpox cows, 4 - link control phage-assistant MC with virus ectromelia, 5 - with vaccinia virus, 6 - smallpox of the cow.

Figure 4. Neutralization of the infectivity of the virus of smallpox, ospowiki and smallpox cows rahovym single-chain antibody 1A and phage-assistant MC. Legend: 1 - neutralization of infectivity of vaccinia virus single-stranded antibody 1A; 2 - neutralization of smallpox cows single-chain antibody 1A; 3 - neutralize variola virus single-stranded antibody 1A; 4 - neutralization of infectivity of vaccinia virus by phage helper MC; 5 - neutralization of smallpox cows phage helper MC; 6 - neutralize variola virus by phage helper MC.

Figure 5. The nucleotide and encoded by its amino acid sequence fragment of plasmid pHEN-1A, encoding a single-chain antibody human 1A as part of a chimeric protein with shell Belko is P3 phage MK.

6. Amino acid sequence single-stranded human antibodies 1A.

7. The General scheme of the structural organization family pHEN-1A (physical map). 1A is a gene that encodes a single-chain antibody human 1A, p3 - the gene encoding the envelope protein P3 phage MK; lac Z promoter Lac operon R, AMPr - gene resistance to ampicillin; amber - cupressinum stop codon TAG amber; 6 His-tag sequence encoding the peptide is UNKNOWN, are some restriction enzymes cut sites.

For a better understanding of the essence of the present invention it is illustrated by the following examples of its implementation.

Example 1. Affinity enrichment obtained library fagbemi antibodies specific to the virus ectromelia.

An aliquot of cell variant libraries of single-chain antibodies person, containing about 1010clones (25 μl), inoculant in 50 ml of medium 2×YT containing 100 μg/ml ampicillin and 1% glucose, and raise with constant stirring at 37°C until OD600=0.5. After that, the obtained culture infecting phage helper MC (New England Biolabs, UK), adding it in the ratio of 1:20 (the number of bacterial cells: the number of phage particles), and incubated for 30 min at 37°C. Then 20 ml of culture precipitated by centrifugation for 10 min at 3300 g. Sediment resuspended in 10 ml of 2×YT containing 100 μg/ml ampicillin and 5 µg/ml kanamycin, and transfer the suspension in 200 ml of 2×YT containing 100 μg/ml ampicillin and 25 μg/ml kanamycin. The culture is incubated overnight with constant stirring at 30°C. the next day cells E. coli precipitated by centrifugation 10800 g, 10 min in a centrifuge "Beckman J2-21", to the supernatant add 1/5 volume of a solution of PEG/NaCl (20% PEG and 2.5 M NaCl), mixed well and within 1 h and incubated on ice. After that, the suspension is centrifuged at 10800 g 30 minutes the precipitate is dissolved in 40 ml of PBS pH 7.2 and add 8 ml of PEG/NaCl, incubated in ice for at least 20 minutes and spend the deposition of bacteriophages in the centrifuge "Beckman J2-21" at 3300 g for 30 minutes. The supernatant together with the remnants of PEG/NaCl carefully removed, and the residue resuspended in 2 ml of sterile PBS. To remove residual bacterial cells phage suspension centrifuged at 3300 g for 10 minutes Yield of phage particles is approximately 1012-1013plaque-forming units (PFU).

Affinity enrichment of the library is carried out in two consecutive rounds. For a first round selection in the wells of 96-hole tablet (metroliner", Russia) adsorb virus ectromelia at a concentration of 100 μg/ml in PBS and incubated overnight at room temperature. The next day after removal of the antigen wells three times washed with PBS and place of nonspecific binding is saturated with 5% dissolve the ohms of skimmed milk powder in PBS buffer for 2 h at 37°C. In addition, a 5% solution of skim milk absorb additional wells as a negative control. After that, the wells three times washed with PBS and each well add 1011The BATTLE ragovoy library in 100 μl of a solution of PBS with 0.1% tween-20. Tablets incubated for 1.5 hours with constant stirring at 37°C, after which the wells are washed 20 times with a solution of PBS with 0.1% tween-20, and then 20 times with PBS.

The elution of bound peroxidase bacteriophages carried out with the help of triethylamine. For this purpose, the wells add 100 ál of 100 mm triethylamine, incubated for 2-3 minutes, stirring constantly, then erwerbende phages transfer 350 ál of 1M Tris-HCl, pH 7.4.

To obtain phage repertoire of cell culture E. coli TG1 in the phase of exponential growth (10 ml for each eluate) infect received rahovym the eluate (400 µl), incubated for 30 min at 37°C, then centrifuged 10 min at 3000 g. Precipitation resuspended in 1 ml of 2×YT and plated on agar plates with the environment 2×YT containing 100 μg/ml ampicillin and 1% glucose. Cup incubated overnight at 30°C. the Grown cells are collected and stored in environment 2×YT containing 30% glycerol at -70°C.

Amplification selected after the first round of bacteriophages carried out as described previously.

The second round of affinity enrichment carried out similarly. The concentration of virus ectromelia reduced to 50 MK is/ml.

The degree of enrichment of the library of antibodies, specifictime to the virus ectromelia, check using indirect ELISA for the ability of populations of phage antibodies obtained from each round of affinity enrichment, to bind the virus ectromelia (figure 1). This virus ectromelia adsorb on 96-well immunological tablets (metroliner", Russia) in the amount of 300 ng per well. Place of nonspecific binding blocked with a solution of 3%BSA (Sigma) in phosphate buffer, pH 7.2. Then in the wells contribute phage antibodies selected, as previously described, diluted PBS buffer containing 0.1% tween, and incubated 1 hour at 37°C. After washing the wells, making anti-M13 mouse serum at a dilution of 1:16000, and then individuai conjugate alkaline phosphatase (Sigma) at a dilution of 1:6000. As a Chromogen use paranitrophenylphosphate. As a negative control using binding phage antibodies with skim milk. The results are shown in figure 1.

Example 2. Selection of clones capable of producing antibodies that bind virus ectromelia.

To obtain monoclonal phage antibodies specific colony of E.coli TG1-rich against virus ectromelia library subcultured with agarized medium in a liquid environment 2×YT containing 100 μg/ml ampicillin and 1%glucose, and grow to a density equal to ,5 in OD 600. Then cells infecting phage helper MC, precipitated by centrifugation at 3300 g for 10 min and resuspended cellular precipitate in the medium containing 100 μg/ml ampicillin and 50 μg/ml kanamycin. Cells cultivated at 30°C under stirring overnight and then precipitated by centrifugation 10800 g, 10 minutes

The supernatant containing phage antibodies examined for the ability to bind to the virus ectromelia method of TYPHUS. Vaccinia virus adsorb to the wells of the 96-hole immunological tablets (metroliner", Russia) at a concentration of 3 μg/ml Designated nonspecific binding blocked with a solution of 3%BSA (Sigma) in phosphate buffer, pH 7.2. Then in the wells contribute supernatant diluted PBS buffer containing 0.1% tween, in a volume ratio of 1:1 and incubated for 1 hour at 37°C. After washing the wells, making anti-MK rabbit serum at a dilution of 1:16000, and then individuai conjugate alkaline phosphatase (Sigma) at a dilution of 1:6000. As a Chromogen use paranitrophenylphosphate. Control of nonspecific binding is the binding of the virus by phage helper MC, not bearing fragments of antibodies on their surface. As a negative control using binding phage antibodies with BSA. The results are shown in figure 2.

Further from supernatant, showing the signal value to the showing in 3 times or more than the control of nonspecific binding and the negative control, secrete phage antibodies using precipitation with a solution of PEG/NaCl and subsequent centrifugation as described in Example 1.

Example 3. Enzyme-linked immunosorbent assay binding phage antibodies 1A viruses ospowiki, smallpox cows and ectromelia.

Serial dilutions of viruses ospowiki, smallpox cows and ectromelia, starting with 600 ng per well, with a step of 1:4 adsorb to the wells of the 96-hole immunological tablets (metroliner", Russia). Place of nonspecific binding blocked with a solution of 3%BSA (Sigma) in phosphate buffer, pH 7.2. Then in the wells contribute phage particles bearing on its surface an antibody 1A, in the amount of 1011PFU per well in PBS buffer containing 0.1% tween, and incubated 1 hour at 37°C. After washing the wells contribute polyclonal anti-MK antibodies rabbit in a dilution of 1:16000, and then individuai conjugate alkaline phosphatase (Sigma) at a dilution of 1:6000. As a Chromogen use paranitrophenylphosphate. The results of the experiments shown in figure 3.

Example 4. The study of the neutralizing activity of phage antibodies 1A.

Antibody serially diluted in PBS buffer with step 1:4, starting with a concentration of 1013phage particles, and mixed with an equal volume of a suspension of variola, ospowiki and smallpox of the cow, diluted to about 300 PFU/ml Mixture inquire the t at 37°C for 1 hour. Aliquots of each mixture is applied to the monolayer of cell line Vero E6 24-hole tablets at 37°C for 1 hour, after which wash. Then the hole is covered with MEM medium containing 2% FBS and 0.24% agarose. Viable cells are stained after 3-4 days after infection, visualize plaques. As a negative control is used bacteriophage assistant MC, not bearing on their surface a single-chain antibodies. The neutralization titer is defined as the dilution of phage antibodies, in which there is a 50%decrease in the number of plaques. The results are shown in figure 4.

From the above it is seen that the obtained fahmida DNA pHEN-1A, containing the gene single-stranded human antibodies 1A, providing under control of the promoter of lactose operon in E. coli cells the synthesis of single-stranded human antibodies 1A exposed on the surface of filamentous bacteriophage M13, able to neutralize variola virus, vaccinia virus and variola virus cows, comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. (figure 5).

Sources of information

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4. Lim APC, Chan C.E.Z., Wong S.K.K., Chan A.H.Y., E.E. Ooi and B.J. Hanson Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library // J. Virol - 2008. - V.5. - R.

5. Clementi N., N. Mancini, L. Solforosi, M. Castelli, M. Clementi, R. Burioni Phage Display-based Strategies for Cloning and Optimization of Monoclonal Antibodies Directed against Human Pathogens // Int J Mol Sci. - 2012. - V. 13(7). - P. 8273-92.

6. Nimmagadda S.V., Aavula S.M., Biradhar N., S. Sula, Lingala R., D. Chandran, Villuppanoor S.A. Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen // Biologicals. - 2012. - V. 40(4). - P.299-308.

7. N.V. tikunova, Kolokolov A.A., town Chepurnov A. A. Recombinant monoclonal human antibodies against Ebola virus. //DAN, 2001, I. 378, No. 4, S. 551-554.

8. Yoon Christmas eve, N.V. Tikunova, Shingareva L.N., Aliyev BECAUSE, Boldyreva E.F., Morozov V.V., swallow A.A., Nekrasov O.V., Polygalov I., Panin A.A., Il A.A., Kirpichnikov M.P., Sandakhchiev PS Full-size recombinant human antibody against vaccinia virus. //Reports of the Russian Academy of Sciences (DAN), 2006, I. 407: No. 5, S. 702-705.

9. N.V. tikunova, Shingareva L.N., Yoon Christmas eve, Morozov V.V., Aliyev BECAUSE, Boldyreva E.F., Nekrasov O.V., Polygalov I., Panin A.A., Kirpichnikov M.P., Il A.A., Sandakhchiev PS Recombinant plasmid DNA pCL37 and pCH37 encoding polypeptides with the properties of light and heavy chains of human antibodies against vaccinia virus, providing together biosynthesis of full-size antibodies against VIR is sa ospowiki in mammalian cells, and recombinant full-size antibody human IgG1 class, interacting with vaccinia virus. RF patent № 2317330. 2008.

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12. Dubrovskaya CENTURIES, N.V. Tikunova, Morozov V.V., Bormotov NI, Laman A.G., Ulitin A.B., Brovko F.A., Belanov E.F., Il A.A. Combinatorial fahmida library of single-chain antibodies person, enriched with antibodies against vaccinia virus, recombinant fahmida DNA pHEN-2A8, containing a unique gene single-stranded human antibodies that can neutralize vaccinia virus and variola virus cows, and synthetic single-chain antibody man 2A8, can neutralize vaccinia virus and variola virus cows. RF patent №2311927. 2005 (prototype).

13. Dubrovskaya CENTURIES, Ulitin A.B., Laman A.G., Gileva I.P., Bormotov NI, A. Ilyichev, Brovko F.A., Shchelkunov S.N., Belanov E.F., N.V. Tikunova Designing combinatorial immune clonetech single-chain antibodies against orthopoxviruses and breeding from her antibodies to recombinant prF30L variola // Molecular biology. - 2007. - V.41, N1. - S. 173-185.

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A means to neutralize variola virus, representing the synthetic single-chain antibody human 1A having the amino acid sequence shown in Fig.6 exposed on the surface of filamentous bacteriophage M13.



 

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SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).

EFFECT: improvement of the method.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: poxvirus includes defective gene I4L and/or F4L and target nucleic acid containing suicide gene. Besides, a composition containing such a poxvirus, its use for obtaining medical preparation and a treatment method of proliferative disease or disease with increased activity of osteoclasts with its application are described. The proposed group of inventions can be used in medicine.

EFFECT: poxvirus of variolovaccine has oncolytic activity.

34 cl, 18 dwg, 5 tbl

FIELD: medicine.

SUBSTANCE: there are presented two recombinant plasmid DNA pFastBac 1 -G2R-dSECRET and pQE-60-TNFR-CrmB-Ind-67 coding TNF-binding CrmB protein domain. Said recombinant plasmid DNA pQE-60-TNFR-CrmB-Ind-67 is designed for transformation in cells of the strain E.coli SG13009[pRep4] - a producer of TNF-binding CrmB BHO protein domain.

EFFECT: presented group of inventions is applicable for preparing drugs used in therapy of severe human diseases caused by hyperproduction of tumour necrosis factor and may be used in medicine.

3 cl, 3 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: chimeric flavivirus contains at least one mutation in a coat protein of chimeric flavivirus, and one or more mutations in (i) 3'-untranslated region (3'-UTR) of a chimeric flavivirus genome and/or (ii) a capside protein of chimeric flavivirus.

EFFECT: chimeric flavivirus shows reduced viscerotropism as compared to mutation-free chimeric flavovirus; virus is completely attenuated.

32 cl, 8 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: displayed polypeptides include at least one unnatural aryl-azide amino acid such as, for example, para-azido-L-phenylalanine, which is incorporated into the phage-displayed hybrid polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA.

EFFECT: invention enables to modify protein in physiological conditions in which phage activity and survival is maintained.

19 cl, 16 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, virology and veterinary. An attenuated recombinant classical swine fever virus (CSFV) is described. Modification is carried out by progressively mutating a portion of the E2 gene of the highly pathogenic strain Brescia. As a result, two to six amino acids on the section 829-837 are replaced with two to six amino acids which are characteristic for the heterologous E2 glycoprotein of the Bovine Viral Diarrhea Virus (BVDV). A classical swine fever vaccine is also obtained based on the obtained attenuated CSFV. The invention can be used in veterinary.

EFFECT: classical swine fever vaccine is obtained.

7 cl, 7 dwg, 4 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns preparing a genetic construct providing a synthesis of p35d recombinant protein in Escherichia coli cells. There are presented: recombinant plasmid DNA pQE-p35d providing the synthesis of p35d recombinant protein of cowpox virus and containing in accordance with physical and genetic map presented on Fig. 2: pQE30 plasmid vector, a fragment coding MRGSHHHHHHG oligopeptice and a fragment of 17 base pairs, coding a fragment of p35 protein of cowpox virus within 1 to 239 amino acid residues (Fig.1a); Escherichia coli XL1Blue/pQE-p35d B-1252 bacterial strain that is a producer of p35d recombinant protein of cowpox virus, containing recombinant plasmid DNA pQE-p35d deposited in the Collection of Bacteria, Bacteriophages and Fungi of FBUN GNTs VB Vector, registration No. B-1252, and p35d recombinant protein of cowpox virus.

EFFECT: solutions may be used to engineer the test systems and to prepare orthopoxvirus split vaccines.

3 cl, 7 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: recombinant plasmid DNA pGEM-Puro-DS-Apo is proposed comprising synthetic apoptin gene flanked by sequences of vaccinia virus genome of island C10L-C12L, and the recombinant strain VVdGF-ApoS24/2 of vaccinia virus producing apoptin.

EFFECT: invention can be used to develop medicinal products to control cancerous diseases.

2 cl, 5 dwg, 1 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: inventions represent synthetic oligonucleotide primers: P1 - 5'-TGGTTACTATTCCATCACCATT-3' (annealing site 11-32 base pairs), P2 - 5'-CGAAACGTCACTTTCGCAAC-3' (annealing site 259-278 base pairs) and method for detection of DNA of infectious anaemia virus of chickens with their help. The method consists in the fact that primers flank the section of the virus genome, which includes CpG islands and VNTR repetitions in the polymerase chain reaction in real time mode. In case of positive reaction, a peak of the melting curve 92C° appears, and when the reaction is confirmed by means of electrophoresis a fragment is visualised in the gel that corresponds to the size of 268 base pairs.

EFFECT: method of diagnostics makes it possible to determine quantitative content of a virus in tissues and may be used to diagnose infectitious anaemia of chickens.

2 cl, 3 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there are presented two recombinant plasmid DNA pFastBac 1 -G2R-dSECRET and pQE-60-TNFR-CrmB-Ind-67 coding TNF-binding CrmB protein domain. Said recombinant plasmid DNA pQE-60-TNFR-CrmB-Ind-67 is designed for transformation in cells of the strain E.coli SG13009[pRep4] - a producer of TNF-binding CrmB BHO protein domain.

EFFECT: presented group of inventions is applicable for preparing drugs used in therapy of severe human diseases caused by hyperproduction of tumour necrosis factor and may be used in medicine.

3 cl, 3 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: set contains species-specific oligonucleotide primer pairs and appropriate fluorescent-marked probes for conducting one-stage instant identification of several human-pathogenic Orthopoxviruses (VARV, MPXV, CPXV and VACV) by means of real-time multiplex PCR.

EFFECT: invention is intended for instant diagnostics of human and animal Orthopoxvirus infections by real-time multiplex PCR.

10 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.

EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.

2 cl, 3 dwg, 1 tbl, 8 ex

FIELD: molecular biology, gene engineering, medicine.

SUBSTANCE: disclosed is method for determination of different orthopoxviruses based on one-step PCR followed by hybridization of obtained fluorescence labeled amplicones with DNA microarray containing discriminating orthopox- and herpesvirus-hybridization probes.

EFFECT: method for express-diagnosis of orthopoxviruses and discrimination thereof from herpesviruses.

3 cl, 14 dwg, 3 tbl, 4 ex

FIELD: biotechnology, in particular gene engineering.

SUBSTANCE: Gene of B9R protein having high homology with extracellular segment of interferon-gamma receptor is isolated by PCR method from Mankeypox virus genome of strain Zaire-96-1-16. Then said protein is cloned in donor plasmid pFastbAC and via site-specific transposition recombinant bacmid is constructed. Said bacmid is used for pest cell transfection to generate target strain.

EFFECT: new drugs for treatment of human diseases associated with hyperproduction of interferon-gamma.

2 cl, 3 dwg, 6 ex

FIELD: biotechnology, protein engineering.

SUBSTANCE: claimed library represents E.coli TGI cells wherein each cell contains fragmid DNA providing biosynthesis of filamentous bacteriophages exposing unique human single-stranded antibody on surface thereof. Also disclosed is recombinant fragmid pHEN-2A8 DNA containing artificial gene of human single-stranded antibody under control of lactose operon promoter providing synthesis of human single-stranded antibody in composition of chimerical protein with membrane pIII protein of M13 bacteriophage in E.coli cells. Also disclosed is method for production of artificial human single-stranded 2A8 antibody by using such fragmid DNA.

EFFECT: fragmid library useful in medicine.

3 cl, 7 dwg, 10 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: biotechnologies.

SUBSTANCE: invention proposes a nanoantibody (a single-domain antibody) specifically bound to hemagglutinin of A-type flu virus H5N2 and suppressing infection of this virus, which is characterised with aminoacid sequence. Besides, a viral, an adenoviral, an adeno-associated and a lentiviral vector for expression of a nanoantibody and a composition for suppression of progress of infection of A-type flu virus H5N2 containing a nanoantibody and a viral vector are described.

EFFECT: invention can be further used in therapy of infection of A-type flu virus H5N2.

6 cl, 12 dwg, 18 ex, 1 tbl

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