Recombinant plasmid dna pcl37 encoding polypeptide with properties of human light chain of antibody against smallpox virus vaccine, recombinant plasmid dna pch37 encoding polypeptide with properties of heavy chain of indicated antibody and their using

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.

EFFECT: valuable medicinal properties of plasmid DNA.

4 cl, 7 dwg, 6 ex

 

The invention relates to biotechnology, in particular genetic engineering, and is designed in vitro recombinant plasmid DNA containing the result of genetic engineering methods of artificial genes for the light and heavy chains of the full sized human antibodies that are created on the basis of variable fragments of light and heavy chains of recombinant antibodies 1F4 from ragovoy library of single-chain antibodies person, and constant genes IgG1 human cytomegalovirus promoter and saiga BGH polyadenylation causing biosynthesis in human cells SOME of 293 T recombinant full-sized human antibodies of the IgG1 class, specifically interacting with vaccinia virus. The affinity constant for derived recombinant antibodies is 3,54·109±0,38·109M-1.

There are currently no effective means of treatment of orthopoxvirus infections. The only way to fight them is vaccination with vaccinia virus. Inoculation ospowiki gives enough protection, but is accompanied by a local inflammatory reaction necrotic nature with subsequent scarring and severe General reaction (fever, malaise, lymphadenitis, etc.) [1, 2]. In addition, oppresive often accompanied the raised serious vaccine-related complications, the likelihood of which is particularly high among persons with reduced immune status [1, 3]. Part of postvaccinal complications can be prevented by administration of human vaccinia immune globulin [4], but this drug roads and inaccessible, and the use of drugs derived from human blood, is always accompanied by a well-known biological risk. Alternative vaccine immunoglobulin could be human monoclonal antibodies (MAB)specific to orthopoxviruses. However, hybridoma technology does not guarantee obtaining stable cell lines.

One of the modern approaches to the design of these µa of variable specific domains of human IgG selected from a combinatorial phage library of human antibodies and constant domains of a human immunoglobulin. To date, such antibodies designed against a number of antigens, including viral agents[5, 6, 7].

Published information about how to obtain full-length human antibodies against orthopoxviruses with constant affinity of about 1·108M-1[8 prototype]. It should be noted that for use in medical practice, it is necessary to select antibodies with a higher affinity. This can reduce therapeutic dose of immunoglobulins that riodic to reduce adverse immune response to the administered drug in patients.

An object of the invention is to obtain two polypeptides with the properties of a full size light and heavy chains of human immunoglobulin forming in mammalian cells, the antibody of the IgG1 class, interacting with vaccinia virus, having a value of affinity constant of more than 1·108M-1.

The problem is solved by constructing two recombinant plasmid DNA, one of which, pcL37, encodes a fusion polypeptide with properties of the light chain of a full-sized human monoclonal antibodies, other, rn, encodes a fusion polypeptide with properties of the heavy chain of a full-sized human monoclonal antibodies. Joint transfection of plasmids pcL37 and RSN human cell line SOME 293 T provides a synthesis of the polypeptide with properties of a full-sized human antibodies of the IgG1 class, specifically interacting with vaccinia virus. The magnitude of the affinity constants obtained for full-length human antibodies 1F4 is 3,54·109±0,38·109M-1.

Transient biosynthesis of target polypeptides is provided by the presence in plasmids pcL37 and RSN cytomegalovirus promoter and polyadenylation site BGH.

The source of genetic material for constructing the target plasmids are the following Genn is engineering the original design:

a) fahmida pHen1F4, containing the gene for human single-stranded antibodies that interact with vaccinia virus [9];

b) plasmid pBVK14a2 and pBVK1-6, containing a leader sequence, respectively, the heavy and light chains of mouse monoclonal antibody MCA F10 [10];

C) plasmid pCIgG1 containing gene WithN1-CN2-CN3 domains of the heavy chain IgG1 human cloned between the sites Ara I and XhoI [11];

g) plasmid pCkap, containing the gene for Kappa-domain light chain of human IgG [11].

Was first established primary sequence of the variable regions of single-chain antibodies in fahmida pHen1F4. Using polymerase chain reaction (PCR) in the presence of appropriate oligonucleotide primers carried out the unification of the leader sequence of the light chain of the MAB F10 and Kappa-domain light chain of human IgG with a variable fragment of a light chain of clone 1F4 and a similar Association leader sequence of the heavy chain of the MAB F10 and constant humanN1-CN2-CN3-IgG1 domains with the variable region of the heavy chain single-chain antibodies 1F4.

Oligonucleotide primers for constructing gene light and heavy chains of human antibodies against vaccinia virus (direction 5′-3′).

1. CCTTCCCTAGGTCGGACTGTGGCTG

2. CTCATGGGTCTTCTGAGCTGACTC

3. CTCAGAAGACCCATGAGCACCAG

4. AAGCTGGCTAGCCACTTCTTAG

5. AAGCTGGCTAGCAGGCAAGG

6. CCTTCGGGCCCTTGGTGGAGGCACTCGAGACGGTGACC

7. CCAAGCACAGGTGCAGCTGGTGGAG

8. CTGCACCTGTGCTTGGGCACTTTG

9. CCATTCAGATCCTCTTCTGA

The Assembly of the gene light chain consists of the following steps:

1. The Association of the variable region of the light chain single-chain antibodies 1F4 with the leader sequence of the light chain of murine antibody F10.

To do this, in the first stage PCR amplified leader sequence, using as matrix pBVK1-6 [10] in the presence of primers 3 and 5, and a variable region light chain single-chain antibodies 1F4 encoded by plasmid pHen1F4, in the presence of primers 2 and 9. Amplification products after separation on agarose combined and used as template for the second stropen PCR in the presence of primers 5 and 9. The resulting fragment containing the variable region leader sequence, is treated with a restriction enzyme XmaJI.

2. Cloning of the gene Kappa-domain light chain of human IgG in plasmid pSK (+) with the simultaneous introduction of website Xma JI 5-end of the gene.

Gene Kappa-domain cloned into plasmid pcDNA3.1 contains a XhoI site at the 3′end. For its Association with the gene coding for the variable region of light chain, built-in fahmida pHen1F4, you must enter the website XmaJI 5′-end since this unique website ends of the variable region. Amplificati is carried out in the presence of primers 1 and BGH (Invitrogen), amplification products treated with restriction enzyme XhoI and inserted into the plasmid pSK (+) sites EcoRV and XhoI. The result is the plasmid pSKCl.

3. Build a full-sized gene light chain.

Plasmid pSKCl treated with restrictase SmaI and XmaJI and unite in the reaction ligating the fragment obtained in step 1. The obtained plasmid pSKl-l transform cells of E. coli. Screening of clones was performed using restriction analysis. Clones containing full-sized light chain gene, analyzed by sequencing. Forth from the thus obtained intermediate plasmids pSKl-l when processing its restriction endonucleases NheI and XhoI get a DNA fragment containing the full-size light chain gene, and embed it in the expression plasmid pcDNA3.1 (+) (Invitrogen), treated the same restrictases. The resulting target recombinant plasmid pcL37 analyze the hydrolysis endonucleases HaeIII, NheI, XmaJI and XhoI. The nucleotide sequence of the gene encoding the hybrid protein was confirmed by sequencing, which was performed by the method and using the set Cycle ReaderTM DNA Sequencing Kit (Fermentas, Lithuania) [12]. The nucleotide sequence of light chain gene and encoded by its amino acid sequence of the recombinant polypeptide is shown in figure 1.

Recombinant plasmid DNA pcL37 encoding synthesis in KL is located mammalian polypeptide with properties of the light chain of a full-sized human monoclonal antibodies, interacting with vaccinia virus, is characterized by the following features:

- has a molecular mass of 4,03 MDA and size 6108 P.O.;

encodes a hybrid protein in which the variable domain light chain single-chain antibodies person 1F4 combined with the constant domain of the Kappa chain of human IgG;

- consists of the following elements:

a) NheI/XhoI vector fragment of plasmid pcDNA3.1(+) (Invitrogen) size 5338 BP containing the promoter is a CMV enhancer, a polyadenylation site, and the site of transcription termination BGH, gene β-lactamase (bla)gene of resistance to neomycin;

b) NheI/XhoI fragment of intermediate plasmids pSKI size 770 BP, containing an artificial gene that encodes a polypeptide with properties of the light chain of a full-sized human monoclonal antibodies that interact with vaccinia virus;

- contains:

a) cytomegalovirus (CMV) promoter and enhancer of transcription;

b) artificial gene that encodes a hybrid protein in which the variable domain light chain single-chain antibodies person 1F4 combined with the constant domain of the Kappa chain of human IgG;

g) a unique recognition sites of restriction endonucleases, with the following coordinates: Bg1II - 12, NheI - 895, XmaJI - 1294, XhoI - 1665.

The Assembly of the gene heavy chain carried out as follows:

1. At the first stage amplified the leader of the th sequence of the heavy chain of the MAB F10 in the presence of primers 8 and 4, and a variable region heavy chain antibodies 1F4, using as matrix fahmida pHen1F4, in the presence of primers 7 and 6 (Ara) with simultaneous introduction of saiga for the restriction enzyme ApaI, which has a 5′-the end of the constant region of IgG1. Amplification products analyzed by agarose gel electrophoresis; the strips of the desired size cut out, and DNA extracted from the gel are combined and used as a matrix for the second stage PCR in the presence of primers 4 and 6. The resulting fragment containing the variable region leader sequence, treated with restriction endonucleases NheI and ApaI, purified by agarose gel electrophoresis and after extraction from agarose gel used in further reactions ligation.

2. Full gene heavy chain receive a three-part legirovaniem fragment obtained in the previous step, with ApaI-XhoI fragment of plasmid pCIgG1 containing the gene encoding WithN1-CN2-CNThe 3 domains of human IgG1, limited sites ApaI and XhoI, with part of the vector plasmid pcDNA3.1 treated with restriction endonucleases NheI and XhoI.

Received target recombinant plasmid RSN analyze hydrolysis by restriction endonucleases HaeIII, NheI, ApaI and XhoI. The structure of the gene encoding a hybrid protein, confirmed by sequencing, which was performed by the method and with what ispolzovaniem set Cycle ReaderTM DNA Sequencing Kit (Fermentas, Lithuania). The nucleotide sequence of heavy chain gene and encoded by its amino acid sequence of the recombinant polypeptide shown in figure 2.

Recombinant plasmid DNA RSN encoding synthesis in mammalian cells polypeptide with properties of the heavy chain of a full-sized human monoclonal antibodies that interact with vaccinia virus, is characterized by the following features:

- has a molecular mass of 4.57 MDA and size 6911 P.O.;

encodes a hybrid protein in which the variable domain of the heavy chain single-chain antibodies person 1F4 combined with the constant domain of the heavy chain of human IgG;

- consists of the following elements:

a) NheI/XhoI vector fragment of plasmid pcDNA3.1 (+) (Invitrogen) size 5338 BP containing the promoter is a CMV enhancer, a polyadenylation site, and the site of transcription termination BGH, gene β-lactamase (bla)gene of resistance to neomycin;

b) ApaI/XhoI fragment of intermediate plasmids pCIgG1 size 1126 BP containing the gene CH1-CH2-CHThe 3 domains of human IgG1;

in) NheI/ApaI fragment size 447 BP, the resulting amplification and encoding the leader sequence of the heavy chain of the antibody F10 and the variable region of the heavy chain of the antibody 1F4;

- contains:

a) cytomegalovirus (CMV) promoter and enhancer TRANS is reply;

b) artificial gene that encodes a hybrid protein in which the variable domain of the heavy chain single-chain antibodies person 1F4 combined with the constant domain of human IgG1;

as genetic markers, gene β-lactamase (bla), which determines the stability of the transformed plasmid RSN cells of bacteria to ampicillin, and the gene of resistance to neomycin for selection of transfected by plasmid RSN of mammalian cells;

unique recognition sites of restriction endonucleases, with the following coordinates: Bg1II - 12, NheI - 895, ApaI - 1338, XhoI - 1320 and 2468.

The invention consists in the following:

- Genetic engineering methods derived plasmids pL37 and RSN, artificial genes encoding light and heavy chains of the full-length human antibodies generated on the basis of variable fragments of light and heavy chains of single-chain antibodies person 1F4, const genes IgG1 human cytomegalovirus promoter and polyadenylation site BGH4.

- Designed genes responsible for the biosynthesis in mammalian cells polypeptide with properties of light and heavy chains of human antibodies, which are combined in antibody class IgG1 directed against vaccinia virus.

To obtain recombinant proteins with the properties of a full size light and heavy chains immune the globulin human, forming an antibody of the IgG1 class, interacting with vaccinia virus, plasmids pcL37 and RSN, transferout SOME T human cells with subsequent separation by affinity chromatography on a column with a carrier Protein G Sepharose (Amersham) in the target protein. The specificity obtained with full-length human antibodies was demonstrated using immunoblot analysis of proteins from vaccinia virus (see example 5). The affinity of the obtained antibodies in binding assays with vaccinia virus determined by ELISA and has the value of the constants 3,54·109±0,38·109M-1that is 30 times higher than in the prototype (see example 6).

The invention is illustrated by the following figures:

Figure 1. The nucleotide sequence and encoded by its amino acid sequence NheI/XhoI fragment of plasmid pcL37 encoding a polypeptide with properties of the light chain of human antibodies against vaccinia virus. Underlined the recognition sites of restriction endonucleases. Initiation and termination codons are shown in bold.

Figure 2. The nucleotide sequence and encoded by its amino acid sequence NheI/XhoI fragment of plasmid RSN encoding a polypeptide with properties of the heavy chain of human antibodies against vaccinia virus. Underlined the recognition sites of restriction endonucleases. The initiator and the term is dominant codons shown in bold.

Figure 3. Physical map of plasmid pcL37. Specified the recognition sites of restriction endonucleases. Pcmv - cytomegalovirus promoter; S, signal sequence; Vl, k - variable and constant parts of the gene light chain recombinant antibodies; BGHpA - site polyadenylation bovine growth hormone.

Figure 4. Physical map of plasmid RSN. Specified sites of restriction endonucleases. Pcmv - cytomegalovirus promoter; S, signal sequence; Vh, const. - variable and constant parts of the gene heavy chain recombinant antibodies; BGHpA - site polyadenylation bovine growth hormone.

Figure 5. Electrophoregram purified target antibodies in a 13% polyacrylamide gel. Tracks: 1 - full size antibody 1F4, not treated with 2-mercaptoethanol; 2 full - length antibody 1F4 treated with 2-mercaptoethanol; 3 - standards of molecular weights.

6. Electrophoregram protein of vaccinia virus, separated by electrophoresis in a denaturing 12% polyacrylamide gel: A - coloring proteins of vaccinia virus, Kumasi R-250 (1), (B) immunoblotting with rivers-ICA 1F4 (2), with the conjugate antivitamin antibodies (anti-human) with alkaline phosphatase (3), M is the molecular mass standards.

7. Determination of affinity constant using indirect enzyme immunoassay. Shows the graph of the titration of the obtained recombinant is nitela. On a solid phase adsorbed vaccinia virus at a concentration of 2.5 µg/ml Recombinant human antibody applied in serial dilutions in increments of 1:3, the original concentration of 5.5 μg/ml, and manifested individuum conjugate of alkaline phosphatase at a dilution of 1/10000. The value of affinity constant was:AF=3,54×109±0,38×109M-1.

For a better understanding of the invention the following specific examples of its implementation.

Example 1. Construction of recombinant plasmid DNA pL37.

a) Combining the variable region of the light chain single-chain antibodies 1F4 with the leader sequence of the light chain of the murine MCA F10.

Amplification of the leader sequence is carried out in a volume of 100 μl. The reaction mixture contains 100 ng pBVK1-6, 50 pmole primers 3 and 5, the dNTP mixture (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 2.5 mM MgSO4,2.5 edict.PfuDNA polymerase (Stratagene) and 1 edict. Taq DNA polymerase (Fermentas). Spend 25 cycles under the scheme: 95°C/40 sec, 45°C/40 sec, 72°C/1 min, the reaction Products analyzed by electrophoresis in 1%agarose gel; lane with a length of about 96 BP cut, DNA extracted from the gel. Amplification of the variable region of the light chain is carried out in a volume of 100 μl in the presence of 100 ng pHen1F4, 50 pmole primers 2 and 9, dNTP mix (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KC, 2.5 mM MgSO4,2.5 edict.PfuDNA polymerase (Stratagene) and 1 edict. Taq DNA polymerase (Fermentas). Spend 25 cycles under the scheme: 95°C/40 sec, 45°C/40 sec, 72°C/1 min, the reaction Products analyzed by electrophoresis in 1%agarose gel; lane with a length of about 360 BP cut, DNA extracted from the gel, together with a previously received fragment and used as matrix in the second stage of PCR, which is carried out in the same conditions in the presence of primers 5 and 9. The amplification product with a length of about 450 BP is treated with a restriction enzyme XmaJI in accordance with the methodology described in [13]. The reaction products analyzed by electrophoresis in 1%agarose gel; a strip of the desired length are cut, the DNA extracted from the gel.

b) Cloning of the gene Kappa-domain light chain of human IgG in plasmid pSK (+) with the simultaneous introduction of website XmaJI 5′-end of the gene.

Gene Kappa-domain amplified in a 100 μl reaction mixture in the presence of 100 ng pCkap, 50 pmole primers 1 and BGH, dNTP mix (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 2.5 mM MgSO4,2.5 edict.PfuDNA polymerase (Stratagene) and 1 edict. Taq DNA polymerase (Fermentas). Spend 25 cycles under the scheme: 95°C/40 sec, 43°C/40 sec, 72°C/1 min, the reaction Products analyzed by electrophoresis in 1%agarose gel; lane with a length of about 440 BP cut, DNA extracted from the gel and education is anywayt the restriction enzyme XhoI. Then, 5 μg of plasmid DNA pSK (+) (Stratagene, USA) treated with restriction endonucleases EcoRV and XhoI, and the reaction products analyzed by electrophoresis with 0.8%agarose gel, the band with the vector DNA cut out and the DNA extracted from the gel. The obtained PCR fragment length 440 BP and the vector portion of the plasmid pSK (+) are combined in a ligation reaction in 20 μl of reaction mixture under standard conditions [13]. 5 μl of the reaction mixture used to transform competent cells XL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 ágml ampicillin. In the process of sieving cells on the surface of agar add 100 ál of 0.1 M solution of IPTG and 20 ml of 4%solution of X-Gal. From growing up white clones secrete plasmid DNA and analyze it by restriction endonucleases EcoRV, XmaJI and XhoI. The nucleotide sequence of the embedded fragment in the selected clones was confirmed by sequencing using the set Cycle ReaderTM DNA Sequencing Kit (Fermentas, Lithuania) as described in the manual [12]. The result is an intermediate plasmid pSKCl.

C) Build a full-sized gene light chain.

5 μg of the plasmid pSKCl treated with restrictase SmaI and XmaJI, the reaction products analyzed by electrophoresis with 0.8%agarose gel. The band with the Linearisation of the vector part of the cut plasmid DNA extracted from the gel and combined in a ligation reaction with 1 μg of the fragment is, obtained in step a) in a 15 μl reaction mixture according to standard methods [13]. 5 μl of the reaction mixture used to transform competent cells of E. coli XL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin. Screening of plasmid DNA from clones conduct restriction fragments length polymorphism analysis using restriction endonucleases XmaJI, NheI and XhoI. Clones containing full-sized light chain gene, analyzed by sequencing as described above. 10 μg of the thus obtained intermediate plasmids pSKl-1 is treated with restriction endonucleases NheI and XhoI, and the reaction products analyzed by electrophoresis in 1%agarose gel, a band with a DNA length of approximately 775 BP cut out and the DNA extracted from the gel. The obtained DNA fragment are combined in a ligation reaction with 1 µg DNA expression plasmid pcDNA3.1 (+) (Invitrogen), treated with the same restriction endonucleases. Transformation and analysis of clones was carried out as described above. The resulting recombinant plasmid pL37 (3) analyze the hydrolysis endonucleases HaeIII, NheI, XmaJI and XhoI. Structure of the gene encoding a hybrid protein, confirmed by sequencing (figure 1).

Example 2. Construction of recombinant plasmid DNA RSN.

a) Association of variable regions of heavy chain antibodies 1F4 with the leader sequence of the heavy chain of murine MCA F10. And is plification leader sequence is carried out in a volume of 100 μl. The reaction mixture contains 100 ng pBVK14a2, 50 pmole primers 8 and 4, dNTP mix (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 2.5 mM MgSO4,2.5 edict.PfuDNA polymerase (Stratagene) and 1 edict. Taq DNA polymerase (Fermentas). Just spend 25 cycles under the scheme: 95°C/40 sec, 45°C/40 sec, 72°C/1 min, the reaction Products analyzed in 1%agarose gel; lane with a length of about 90 BP cut, DNA extracted from the gel. Amplification of the variable region of the heavy chain is carried out in a volume of 100 μl in the presence of 100 ng pHen1, 50 pmole primers 6 and 7, dNTP mix (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 2.5 mM MgSO4,2.5 edict.PfuDNA polymerase (Stratagene) and 1 edict. Taq DNA polymerase (Fermentas). Spend 25 cycles under the scheme: 95°C/40 sec, 45°C/40 sec, 72°C/1 min, the reaction Products analyzed by electrophoresis in 1%agarose gel; lane with a length of about 390 BP cut, DNA extracted from the gel, together with a previously received fragment and used as matrix in the second stage of PCR, which is carried out in the same conditions in the presence of primers 4 and 6. The amplification product with a length of about 465 BP treated with restriction endonucleases NheI and ApaI, and the reaction products analyzed by agarose gel electrophoresis. Strip approximately 460 BP cut, DNA extracted from the gel.

b) Build a full-sized heavy chain gene. 10 μg of plasmid pCIgG1 process restrict the tears ApaI and XhoI, the reaction products analyzed by electrophoresis in 1%agarose gel. The band length of about 1130 BP, containing the gene for CH1-CH2-CHThe 3 domains of human IgG1 cut, DNA extracted from the gel. Then, 5 μg of the plasmid pcDNA3.1 (+) (Invitrogen) treated with restriction endonucleases NheI and XhoI, and the reaction products analyzed by electrophoresis in 0.8%agarose gel, the band with the Linearisation of the vector part of the cut plasmid DNA extracted from the gel. Next 1 MGK ApaI-XhoI fragment, 1 µg. the linearized vector part and 0.5 μg PCR fragment obtained in step (a), combine in a ligation reaction in 20 μl according to standard methods [13]. 5 μl of the reaction mixture used to transform competent cells of E. coli XL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin. Screening of clones conduct restriction fragments length polymorphism analysis using the restriction endonucleases ApaI, NheI and XhoI. The resulting recombinant plasmid RSN (Fig. 4) analyze the restriction endonucleases HaeIII, NheI and XhoI. Clones containing full-sized heavy chain gene, analyzed by sequencing as described above.

Example 3. The full expression of human antibodies in cell lines of human rights.

Transient expression of full-size human antibodies against vaccinia virus is carried out in the result t is inspectie human cell lines NEXT, the obtained expression plasmids containing the genes for the light and heavy chains of antibodies. To do this, before transfection cells NECT cultivated at 37°C in an atmosphere of 5% CO2in DMEM (Gibco BRL)containing 10% fetal bovine serum. Cells plated on 60 mm plates and grown to a density of 90-95%, followed by transfection.

The transfection is carried out as follows. Ten micrograms of plasmid DNA (5 μg DNA plasmid pcL37 and 5 μg DNA plasmid pcH37) was diluted in 500 μl of DMEM medium. Simultaneously mix 25 ál of a solution of LipofectAmine 2000 Reagent (Gibco BRL) c 500 ál of DMEM medium. Both of the obtained solution are combined and incubated for 20 min at room temperature. After incubation, the solution of DNA-LipofectAmine bring in DMEM and the resulting mixture was used as culture medium for cells NEXT.

After transfection cells grown for 72 h without replacing the culture medium. Upon completion of incubation, the culture medium is collected and centrifuged 5 min at 1,500 rpm. The supernatant was separated and used for the selection of antibodies.

Example 4. The selection of recombinant antibodies using affinity chromatography.

150 ml of the culture fluid is applied to a column containing 1 ml of carrier Protein G Sepharose (Amersham), equilibrated with PBS buffer. The column was washed with 10 ml of the same buffer and recombinant protein elute with 0.1 M glycine-Cl, the pH of 2.7, collecting fractions of 0.5 ml each faction immediately add 50 ál of 2 M Tris-HCl, pH 8.0 to neutralize. Fractions containing recombinant protein, analyzed by electrophoresis in polyacrylamide gel, combine and cialiswhat against PBS buffer. Purified recombinant antibodies characterized by fractionation in 12% SDS page with SDS, preparing samples for application to the gel in the presence and absence of 2-mercaptoethanol (figure 5).

Example 5. The specificity of the obtained recombinant antibodies confirmed using immunoblot analysis of proteins from vaccinia virus (6). Proteins of vaccinia virus separated by electrophoresis in 12% polyacrylamide gel with SDS, transferred to a nitrocellulose filter of 0.45 μm ("Millipore, USA), which after blocking of nonspecific binding sites with a solution of 3% BSA (Sigma, USA) incubated with the test antibody. Bound peroxidase antibodies detected by adding alkaline phosphatase conjugate (Sigma, USA) at a dilution of 1:10000. Visualizza immune complex is carried out by adding 5-bromo-3-indole phosphate (Carl Roth GmbH, Germany) and nitrotetrazolium blue (Sigma, USA). The results show binding specificity designed with full-length antibody with a protein of about 27 kDa of vaccinia virus (6).

Example 6. Determination of affinity constant of the obtained recombinant for sorazmerno antibodies in binding assays with vaccinia virus. The affinity constant determined by ELISA. Solid phase adsorb viral drug at a concentration of 250 ng/well, and after locking the places nonspecific binding solution of 2% bovine serum albumin (BSA, Sigma, USA) incubated with the obtained recombinant antibody added in serial dilutions, starting with a concentration of 5.5 μg/ml increments dilution 1:3. The immune complex is detected by adding individualo conjugate alkaline phosphatase (Sigma, USA) at a dilution of 1:10000. As a Chromogen used pair-nationalpost (ICN, USA). As a negative control in parallel experiments testing the binding of the obtained recombinant antibodies with BSA. The experimental results show the ability of the obtained recombinant antibodies to bind the vaccinia virus and the lack of interaction between the obtained antibody and BSA. The value of affinity constant is calculated by the method of least squares according to the formula:

where x is the concentration of antibody in solution, R is the concentration of adsorbed antigen, k is the affinity constant, And is the normalization factor. The value of constant affinity for recombinant human antibodies 1F4 is 3,54×109±0,38×109M-1(Fig.7). Such an antibody can be considered to Vysochin the M.

Thus, the constructed plasmids containing synthetic genes for the light and heavy chains of the full sized human antibodies that are created on the basis of variable fragments of light and heavy chains of single-chain antibodies person 1F4, const genes IgG1 human cytomegalovirus promoter and polyadenylation site BGH4. Transient transformation of eukaryotic cell line NEXT leads to the biosynthesis of polypeptides with the properties of light and heavy chains of human antibodies, which are combined in the antibody of the IgG1 class, specifically interacting with a protein of 27 kDa of vaccinia virus, with KAF=3,54×109±0,38×109M-1that is 30 times higher than in the prototype.

Received a full-size recombinant antibody against the protein of 27 kDa of vaccinia virus can serve as a basis for the creation of pharmaceutical products for the diagnosis and treatment of a number of post-vaccination complications caused by vaccinia virus. In this case, preparations will contain a reduced therapeutic doses of immunoglobulins, which will minimize unwanted immune response to the administered drug in patients.

Sources of information

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6. Kausmally L., K. Waalen, Lobersli I., Hvattum, E., Bemtsen G., Michaelsen T., Brekke O.H. Neutralizing human antibodies to varicella-zoster virus (VZV) derived from a VZV patient recombinant antibody library. J. Gen. Virol. 2004. Vol.85. P.3493-3500.

7. Williamson R.A., R. Burioni, Sanna P.P., L.J. Partridge, Barbas CF, Burton D.R. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc. Natl. Acad. Sci. 1993. Vol.90. P.4141-4145

8. International application WO No. 03068151, CL SC 16/08, publ. 21.08.2003.

9. Morozov V.V., N.V. Tikunova, Batanova T.A., Yoon Christmas eve, Bowszyc H., Jirkovsky E.V., V.V. Voronin, Bobko DI, Belanov E.F., Il A.A., Sandakhchiev PS Mini-human antibodies against orthopoxviruses. Herald of the Russian Academy of medical Sciences, 2004, №8. s-27.

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14. Varfolomeev S.D., Gurevich KG of Biokinetic: a Practical course. M: fair-PRESS, 1999.

1. Recombinant plasmid DNA pL37 encoding synthesis in mammalian cells polypeptide with properties of the light chain of the full-size monoclonal antibodies of the person interacting with the vaccinia virus, the size 6108 softwares molecular weight 4,03 MDA, including:

NheI/XhoI vector fragment of plasmid pcDNA3.1(+) (Invitrogen) size 5338 BP, containing the cytomegalovirus (CMV) promoter and enhancer of transcription, polyadenylation site and the site of transcription termination BGH, gene β-lactamase (bla) and the gene of resistance to neomycin;

NheI/XhoI - fragment 770 BP, containing an artificial gene that encodes a hybrid polypeptide in which the variable domain light chain single-chain antibodies 1F4 obtained from combinatorial ragovoy library of single-chain antibodies person is connected with a constant domain Kappa-chain IgG person having the nucleotide sequence shown in figure 1;

genetic markers:

gene β-lactamase (bla), which determines the stability of the transformed plasmid pcL37 cells of bacteria to ampicillin;

the gene of resistance to neomycin for selection of transfected by plasmid pL37 leakmalaysia;

unique recognition sites of restriction endonucleases, with the following coordinates: Bg1II - 12, NheI - 895, XmaJI - 1294, XhoI - 1665.

2. Recombinant plasmid DNA RSN encoding synthesis in mammalian cells polypeptide with properties of the heavy chain full-size monoclonal antibodies of the person interacting with the vaccinia virus, the size 6911 softwares molecular weight of 4.57 MDA, including:

NheI/XhoI vector fragment of plasmid pcDNA3.1(+) (Invitrogen) size 5338 BP, containing the cytomegalovirus (CMV) promoter and enhancer of transcription, polyadenylation site, the site of termination of transcription BGH, gene β-lactamase (bla) and the gene of resistance to neomycin;

ApaI/XhoI - fragment of intermediate plasmids RDS size 1126 BP containing the gene Withn1-Cn2-CnThe 3 domains of human IgG1;

NheI/ApaI fragment size 447 BP, containing an artificial gene that encodes a leader sequence of the heavy chain of the antibody F10 and the variable region of the heavy chain single-chain antibodies 1F4 obtained from combinatorial ragovoy library of single-chain antibodies person having the nucleotide sequence shown in figure 2;

genetic markers:

gene β-lactamase (bla), which determines the stability of the transformed plasmid RSN of bacterial cells to any illino;

the gene of resistance to neomycin for selection of transformed plasmid RSN of mammalian cells;

unique recognition sites of the restriction endonucleases, with the following coordinates: Bg1II - 12, NheI - 895, ApaI - 1338, XhoI - 1320 and 2468.

3. The use of recombinant plasmid DNA pL37 and RSN, characterized in claims 1 and 2, to obtain the recombinant full-length human antibodies of class IgGI, interacting with vaccinia virus, having a constant affinity of 3.54·109±0,38·109M-1, through co-transfection with the indicated plasmid DNA of human cells SOME T.



 

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FIELD: medicine, immunology.

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FIELD: gene engineering.

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FIELD: biotechnology, immunology.

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FIELD: biotechnology, immunology, medicine, oncology.

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103 cl, 169 dwg, 6 tbl, 30 ex

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EFFECT: valuable medicinal properties of peptide.

16 cl, 2 tbl

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FIELD: immunology, medicine.

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45 cl, 14 tbl, 28 ex

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EFFECT: valuable biological and medicinal properties of antibodies.

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FIELD: immunology, biotechnology, in particular antibody modified by gene-engineering methods.

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EFFECT: antibody fragments with improved specificity to human cancer-embryonic antigen.

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FIELD: immunology, biotechnology.

SUBSTANCE: described are rabies virus-neutralizing antibody and fragments thereof; method for treatment of subject being affected by rabies virus using said antibody and fragments thereof. Disclosed are variants of isolated nucleic acids, encoding polypeptides bearing light and/or heavy antibody strain, respectively. Disclosed is expression vector bearing at least one from abovementioned nucleic acids. The invention makes it possible to increase subject survival after rabies virus attack.

EFFECT: method for prophylaxis and treatment of rabies virus infections.

14 cl, 1 tbl, 5 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

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