Recombinant plasmid dna pclm4/hygro-14d5 coding polypeptide with light chain properties of chimeric antibody to tick-borne encephalitis virus, and recombinant plasmid dna pchm2-14d5 coding polypeptide with heavy chain properties of chimeric antibody to tick-borne encephalitis virus, chimeric antibody providing acute prevention of tick-borne encephalitis in mice

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology, gene and protein engineering. There are engineered plasmids pCLm4/hygro-14D5 and pCHm2-14D5. The plasmids provide the synthesis in eukaryotic cells of polypeptides with light and heavy chain properties of a chimeric antibody, which are combined into the chimeric antibody ch14D5 class IgG1/kappa. The producing chimeric antibody ch14D5 recovered from the culture fluid of CHO-K1 cells transfected by plasmids pCLm4/hygro-14D5 and pCHm2-14D5 having a molecular weight of about 150 kDa. The antibody consists of two identical light chains, a constant portion of which corresponds to human kappa antibodies, and two identical heavy chains, a constant portion of which corresponds to human IgGI antibodies; having an amino acid sequence SEQ ID NO: 1 and SEQ ID NO: 2.

EFFECT: antibody is targeted to tick-borne encephalitis and provides the acute prevention of mice against tick-borne encephalitis.

3 cl, 7 dwg, 8 ex

 

Group of inventions relates to biotechnology, in particular genetic engineering, and is intended for biosynthesis in mammalian cells, full-sized chimeric antibodies, which provides emergency preventing mice from viral encephalitis.

Tick-borne encephalitis virus, member of the family Flaviviridae, is highly pathogenic to humans viral agent that can cause disease, resulting in serious lesions of the nervous system [1].

Now for etiotropic therapy of tick-borne encephalitis used mainly serum immunoglobulin manufactured from plasma of blood donors living in natural foci of the disease [2]. This drug has a pronounced therapeutic effect especially with moderate and severe disease, and the introduction of the drug in 1-2 days after the bite produces a much greater therapeutic effect than the introduction in the following days. However, the drug has certain drawbacks, in particular, it is scarce due to limited source of the original material, and contains a relatively low level of specific neutralizing antibodies. In addition, the use of drugs for human blood is accompanied by known biological risk.

In recent years, whey �ntitle replace recombinant antibodies, obtained in cell cultures. Among the recombinant antibodies in the pharmaceutical market is dominated by the chimeric antibody is a full - size antibodies in which the constant domains of human immunoglobulins attached to the variable domains of murine monoclonal antibodies (MABs) [3, 4]. When constructing chimeric antibodies against tick-borne encephalitis virus key step is selecting the ICA, the variable domains of which have high affinity for the antigen and obepechivaet Wysokie antiviral properties.

Known analogs of vector plasmids used for expression of chimeric antibodies and fully human antibodies. Such analogs include plasmid DNA RSN and pCL37 (patent RU 2317330 C2, op. 20.02.2008) designed for expression of human antibodies against the vaccinia virus; plasmid DNA RSN and pCL1 (patent RU 2285043 C2, op. 10.10.2006) designed for expression of human antibodies against the Ebola virus; plasmid DNA pH1-HD and pL-HD2 (patent US 4975369 A, op. 04.12.1990 and SU 1780541 A3, op. 07.12.1992) designed for expression of a chimeric antibody KS1/4 antigen against EP-Cam adenocarcinoma cells human; plasmid DNA pSVgptHu2VHPLAP-HuIgG1 and pSVneoHuVkPLAP-HuCk, designed for expression of humanized antibody against placental alkaline phosphatase (patent WO 199107500 A1, op. 30.05.1991 and patent RU 2102479 C1, op. 2.01.1998). Information about plasmid DNA intended for the production of chimeric or humanized antibodies against tick-borne encephalitis virus, publicly available sources of information are missing.

Were previously obtained and characterized μa against glycoprotein E - the main immunogenic protein of tick-borne encephalitis virus, and some of them, in particular 14D5) antibody capable of inhibiting viral infectivity in the culture of eukaryotic cells [5].

The closest analogue to the claimed group of inventions is the prototype, is a chimeric antibody ch13D6, with affinity to the glycoprotein E of tick-borne encephalitis virus with an affinity constant of 1.4·10-8M and capable of neutralizing the infectivity of tick-borne encephalitis virus in cell culture [6]. Antibody ch13D6 get in the expression system based on the eukaryotic line NEXT and two plasmid DNA pD6H and pD6L containing the genes for the heavy and light chains of the chimeric antibody human/mouse class IgG1-Kappa. However, the resulting chimeric antibody is not capable of protecting the animal model of infection by tick-borne encephalitis virus.

The task of the group of inventions is getting two polypeptides with the properties of a full-sized light and heavy chains of human immunoglobulin, forming in mammalian cells, the chimeric antibody human/mouse �Lassa IgG1/Kappa, having higher affinity to the glycoprotein E of tick-borne encephalitis virus, as well as providing emergency preventing mice from tick-borne viral encephalitis in a dosage not exceeding 1 mg per kg of animal weight.

The technical result of the receipt of the glycoprotein immunoglobulin type, which provides emergency preventing mice from tick-borne viral encephalitis in a dosage not exceeding 1 mg per kg of animal weight.

This result is achieved by constructing two recombinant plasmid DNA, one of which, pCLm4/hygro-14D5 encodes the synthesis of the polypeptide with properties of light chain full-sized chimeric antibodies, other, pCHm2-14D5, encodes the synthesis of the polypeptide with properties of the heavy chain of the chimeric antibody. Joint transfection of plasmids pCLm4/hygro-14D5 and pCHm2-14D5 eukaryotic cell line Cho-K1 provides the synthesis of the polypeptide with properties of the chimeric antibody, which provides emergency preventing mice from viral encephalitis. Transient biosynthesis of target polypeptides is provided by the presence of plasmid DNA pCLm4/hygro-14D5 and pCHm2-14D5 cytomegalovirus promoter and polyadenylation site of the gene of bovine growth hormone.

The essence of the claimed group of inventions is as follows.

Genetic engineering techniques have the plasmid pCLm4/hygro-14D5 � pCHm2-14D5, which contain artificial genes of the light and heavy chains of the full-sized chimeric antibodies are created on the basis of the variable fragment of light and heavy chains of murine monoclonal antibody 14D5 against tick-borne encephalitis virus and the genes encoding the constant domains of human immunoglobulin class IgG1/Kappa, and also contain the cytomegalovirus promoter and the polyadenylation site of the gene of bovine growth hormone. The constructed plasmid DNA with the introduction in eukaryotic cells responsible for the biosynthesis of polypeptides with the properties of light and heavy chains of human antibodies, which are combined in a chimeric antibody directed against tick-borne encephalitis virus and provide emergency prevention mice from viral encephalitis.

The source of genetic material for constructing the target plasmids are genetically engineered original design:

a) total RNA isolated from hybrid cells producing the murine monoclonal antibody 14D5 [5];

b) total RNA isolated from total cellular fraction of blood of a healthy donor;

b) plasmid DNA pCL37, containing the gene for the light chain of full-size monoclonal antibodies 1F4 against vaccinia virus, comprising the leader sequence of the gene light chain ant�tel mouse the plot of the gene encoding the constant domain of the light chain of human antibodies of class Kappa [7];

d) plasmid DNA RSN, containing the gene for the heavy chain of full-size monoclonal antibodies 1F4 against vaccinia virus, comprising the leader sequence of the heavy chain gene of the mouse antibody, the sites of genes coding constant CH1-CH2-CH3 domains of the heavy chains of human antibodies of class IgG1 [7];

e) plasmid DNA pcDNA™3.1/Hygro(+) (Invitrogen);

(e) the oligonucleotide primers for constructing plasmid

DNA containing the genes for the light and heavy chains of antibodies against

tick-borne encephalitis virus (in the direction 5'-3').

1. 5' CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TC

2. 5' GGA AGA TCT ATA GAC AGA TGG GGG TGT CGT TTT GGC

3. 5' CC GAA TTC GAY ATT GTG MTS ACM CAR WCT MCA

4. 5' GG GAA GCT TGA TAC AGT TGG TGC AGC ATC AGC

5. 5' GGC TTA TCG AAA TTA ATA CG

6. 5' TAG AAG GCA CAG TCG AGG

7. 5' GAC AAA ACT CAC ACA TGC

8. 5' - CAT GAG GGT GTC CTT GGG

9. 5' GAA ATC AAA CGT ACT GTG GCT GCA

10. 5' - TGC AGC CAC AGT ACG TTT GAT TTC

11. 5' GG GAT ATC GTG ATG ACC CAG TCC CC

12. 5' CCG TTT GAT CTC AAG CTT GGT CCC

13. 5' GAG GTG CAG CTG CTC GAG TCT GG

14. 5' GAC GGT GAC CAG GGT ACC CTG GCC

15. 5' CGC TCT AGA TCA TTT ACC CGG GGA CAG GGA GAG

16. 5' A TGC AAG CTT GAG ATC AAA CGG ACT GTG GCT GCA CCA TCT GTC

17. 5' TCG AGG ATC CCT AAC ACT CTC CCC TGT TGA AGC

18. 5' CAG GGT ACC CTG GTC ACC GTC TCC TCA GCC TCC ACC AAG GGC CCA T

19. 5' A TGC TCT AGA TCA TTT ACC CGG GGA CAG GGA

The resulting plasmid pCLm4/hygro-14D5 that encodes the synthesis in the cells of melicope�ment of the polypeptide with properties of light chain full-sized chimeric antibody, characterized by the following features:

- has a molecular weight of 4.11 MDA and size 6232 BP;

- encodes a hybrid protein in which the variable domain light chain mouse monoclonal antibody 14D5 is connected to the constant domain of the Kappa chain of human immunoglobulins;

- consists of the following elements:

a) NheI / ApaI - vector fragment of plasmid pcDNA™3.1/Hygro(+) (Invitrogen) 5491 BP containing the promoter-enhancer of cytomegalovirus and the polyadenylation site and the site of termination of transcription of the bovine growth hormone gene β-lactamase (bla) gene hygromycin phosphotransferase (hpt);

b) NheI / ApaI fragment of intermediate plasmids pCLm4-14D5 size 741 BP, including an artificial gene encoding a polypeptide with properties of light chain full-sized chimeric antibodies, engaged in the protection against tick-borne encephalitis virus.

- contains:

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

b) an artificial gene encoding a hybrid protein in which the variable domain light chain mouse monoclonal antibody 14D5 is connected to the constant domain of the Kappa chain of human immunoglobulins;

b) as a genetic marker gene β-lactamase (bla), which determines the stability of the transformed plasmid pCLm4/hygro-14D5 cells of bacteria to ampicillin and the gene of hygromycin posttrans�erase (hpt), providing resistance to hygromycin In for breeding transfected with plasmid pCLm4/hygro-14D5 mammalian cells;

g) a unique recognition sites of the restriction endonucleases, with the following coordinates: BglII - 13, NheI - 896, EcoRV - 980, HindIII - 1284, ApaI - 1637.

The resulting plasmid pCHm2-14D5 that encodes the synthesis in mammalian cells polypeptide with properties of full-size heavy chain of the chimeric antibody, characterized by the following features:

- has a molecular weight 4,47 MDA and size 6765 BP;

- encodes a hybrid protein in which the variable domain of the heavy chain of murine monoclonal antibody 14D5 is connected to the constant part of the heavy chain of human immunoglobulin class IgG1;

- consists of the following elements:

a) NheI / XbaI vector fragment of plasmid pcDNA™3.1 (+) (Invitrogen) size 5332 BP containing the promoter-enhancer of the cytomegalovirus (CMV), a polyadenylation site and the site of termination of transcription of a gene of bovine growth hormone (BGH), the gene for β-lactamase (bla) gene for resistance to neomycin (neo);

b) NheI / XhoI fragment of intermediate plasmids pCHm2 size 104 BP containing the leader sequence of one of VH-genes of the mouse;

b) XhoI / Acc65I fragment size 312 BP, encoding a variable domain of a murine monoclonal antibody 14D5;

g) Acc65I / XbaI fragment of intermediate plasmids pCHm, which includes the segment encoding the CH1-, CH2-, CH3 domains of a human immunoglobulin of the IgG1 class.

- contains:

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

b) an artificial gene encoding a hybrid protein in which the variable domain of the heavy chain of murine monoclonal antibody 14D5 is connected to the constant part of the heavy chain of immunoglobulin IgGI person;

b) as a genetic marker gene β-lactamase (bla), which determines the stability of the transformed plasmid pCHm2-14D5 cells of bacteria to ampicillin and the gene of resistance to neomycin (neo) for selection of transfected the plasmid pCHm2-14D5 mammalian cells;

g) a unique recognition sites of the restriction endonucleases, with the following coordinates: BglII - 14, NheI - 897, XhoI - 1001, Acc65I - 1313, XbaI - 2330.

Transient expression of chimeric antibodies ch14D5 carried out in mammalian cell lines Cho-K1 transfected plasmids simultaneously pCLm4/hygro-14D5 and pCHm2-14D5 for synthesis of polypeptides with the properties of a full-sized light and heavy chains of the chimeric antibody, which provides emergency preventing mice from viral encephalitis.

Target chimeric antibody ch14D5 isolated from the culture fluid affinity chromatography sorbent with covalently immobilized protein A of Staphylococcus aureus. Apportionment�Noah chimeric antibody analyzed by gel electrophoresis in denaturing conditions in the presence and in the absence of 2-mercaptoethanol.

The obtained chimeric antibody ch14D5 has the ability to bind the surface protein E of tick-borne encephalitis virus [8] with an affinity constant of 6.0·10-11M, which is determined by surface plasma resonance using the device ProteOn XPR36 (Bio-Rad).

The obtained chimeric antibody ch14D5 entered in a dosage not exceeding 1 mg per kg of animal weight, provides emergency preventing mice from tick-borne viral encephalitis, which was confirmed by experiments in vivo.

Thus, first obtained plasmid DNA pCLm4/hygro-14D5 and pCHm2-14D5 for synthesis of polypeptides with the properties of a full-sized light and heavy chains of the chimeric antibody capable of binding surface protein E of tick-borne encephalitis virus with an affinity constant of 6.0·10-11M and to provide emergency prophylaxis mice from tick-borne viral encephalitis when administered chimeric antibody ch14D5 in a dosage not exceeding 1 mg per kg of animal weight.

The invention is illustrated by the following figures:

Fig. 1. Nucleotide and encoded by its amino acid sequence of the NheI/ApaI fragment of plasmid pCLm4/hygro-14D5 that encodes the polypeptide with properties of light chain chimeric antibodies against tick-borne encephalitis virus. Underlined the recognition sites of restriction endonucleases, bold PE�and following the termination codon.

Fig. 2. Nucleotide and encoded by its amino acid sequence of the NheI/XbaI fragment of plasmid pCHm2-14D5 that encodes the polypeptide with properties of chimeric heavy chain antibodies against tick-borne encephalitis virus. Underlined the recognition sites of restriction endonucleases bold the initiating and terminating codons.

Fig. 3. Map of plasmid pCLm4/hygro-14D5. Specified sites of restriction endonucleases. Pcmv - cytomegalovirus promoter; S, signal sequence; VL, kappa_const - variable and constant parts of the gene light chain recombinant antibody; BGHpA - the polyadenylation site of the gene of bovine growth hormone.

Fig. 4. Map of plasmid pCHm2-14D5. Specified sites of restriction endonucleases. Pcmv - cytomegalovirus promoter; S, signal sequence; VH, IgG1_const. - variable and constant portions of the gene of the heavy chain of a recombinant antibody; BGHpA - site polyadenylation bovine growth hormone.

Fig. 5. The èlektroforegramme purified chimeric antibody in a denaturing 15% polyacrylamide gel. Lanes: 1 - molecular mass marker, 2 - target protein not treated with 2-mercaptoethanol, 3 - target protein treated with 2-mercaptoethanol.

Fig. 6. A graph illustrating the kinetics of binding of the chimeric antibody ch14D5 in various concentrations with recombinant protein E [8]. Fragment 1-600 sec - phase svyazyvanie� antibodies, 600-2500 h - phase dissociation of the antibody.

Fig. 7. A graph illustrating the survival of mice infected with tick-borne encephalitis virus, strain "Absettarov", in a dosage of 240 LDO, depending on the introduction of the chimeric antibody ch14D5. The horizontal axis shows the days after infection, and the vertical the number of living mice.

For a better understanding of the essence of the proposed invention, they are illustrated by the following examples of a specific implementation.

Example 1. Obtaining DNA fragments encoding the variable domains of murine monoclonal antibody 14D5, and determination of their nucleotide sequences.

Of hybrid cells producing monoclonal antibody 14D5, isolated total RNA by any available method, for example, using "RNeasy Mini Kit" (Qiagen).

On the basis of the selected RNA in the reverse transcription reaction (FROM) -polymerase chain reaction (GSR) using oligonucleotide primers 1 and 2 and set the "One step RT-PCR Kit (Qiagen) is prepared DNA fragment VH-14D5 encoding variable domain of the heavy chain of murine monoclonal antibody 14D5, and similarly with the use of oligonucleotide primers 3 and 4 receive the DNA fragment VL-14D5 encoding variable domain light chain mouse monoclonal antibody 14D5. Products FROM RT-PCR separated using electrophoretic agarose�th gel and eluted from the gel in any way, for example, with the use of the kit GeneJET™ Gel Extraction Kit (Fermentas).

The nucleotide sequence of the obtained DNA fragments of VH-14D5 and VL-14D5 determined by any available method, for example, using the kit BigDye® Terminator v3.1" (Applied Biosystems) and an automated capillary sequencer ABI 3730XL Genetic Analyser (Applied Biosystems). The nucleotide sequence of DNA fragments of VH-14D5 and VL-14D5 encoding the variable domains of the heavy and light chains of murine monoclonal antibody 14D5 shown in Fig. 1 and Fig. 2.

Example 2. Obtaining DNA fragments encoding the constant domain of the heavy chain of human immunoglobulin of the IgG1 class and the constant domain of the light chain of human immunoglobulin of class Kappa.

From 1 ml of blood of a healthy donor is isolated total RNA by any available method, for example, using "RNeasy Mini Kit" (Qiagen).

On the basis of the selected RNA in the reaction RT-PCR using oligonucleotide primers 16 and 17 and a set of "One step RT-PCR Kit (Qiagen) is prepared DNA fragment IGKC encoding the constant domain of the light chain of class Kappa human immunoglobulin. The procedure is performed according to the manufacturer's instructions, a primer annealing is carried out at 55°C. Similarly, using oligonucleotide primers 18 and 19 receive the DNA fragment IGHG1 encoding the constant domain of tulassay human immunoglobulin of the IgG1 class. Products FROM RT-PCR separated by electrophoresis using 1% agarose gel and excise the gel fragment with DNA corresponding to the higher mobility of 1000 BP in the case of IGHG1 and 340 BP in the case of IGKC. Eluted DNA from the gel in any way, for example, with the use of the kit GeneJET™ Gel Extraction Kit (Fermentas).

The nucleotide sequence of the obtained DNA fragments IGHG1 and IGKC determined by any available method, for example, using the kit BigDye® Terminator v3.1" (Applied Biosystems) and an automated capillary sequencer ABI 3730XL Genetic Analyser (Applied Biosystems). The nucleotide sequence of the DNA fragments IGHG1 and IGKC encoding the constant domains of the heavy chain of human immunoglobulin class IgG1 and the light chain of human immunoglobulin of class Kappa is shown in Fig. 1 and Fig. 2.

Example 3. Construction of plasmid DNA pCLm4/hygro-14D5 containing a gene that encodes the light chain of the chimeric antibody ch14D5.

Construction of plasmid DNA pCLm4/hygro-14D5 consists of several stages:

1. Modification of the original plasmid pCL37 for the purpose of unique recognition sites of restriction endonucleases. For this purpose, plasmid DNA hydrolyzing the endonucleases BamHI and XbaI, complete 5'-protruding ends of the vector fragment to blunt using Pfu polymerase and combine the resulting ends using DNA ligase of bacteriophage T4. The obtained intermediate�face-to-face plasmid pCL37deltal used as template in PCR, which is carried out in 50 µl reaction mixture containing 0.5 units of the act. Taq DNA polymerase (Fermentas) and 0.5 units act. Pfu DNA polymerase (Fermentas), 12.5 pmol of each oligonucleotide, 0.2 mm of each deoxynucleotide, 75 mm Tris-HCl (pH 8.8 at 25°C), 20 mm (NH4)2SO4That 0.01% of the detergent tween-20, 2.5 mm MgSO4. Spend 30 cycles according to the scheme: 95°C/30 sec, 52°C/30 sec, 72°C/50 sec. While using a pair of primers 5 and 10, and a pair of primers 6 and 9 get two overlapping DNA fragment, which after separation is used in the reaction of the joint combining PCR with the formation of a single DNA fragment light_delta2, which is a fragment of the plasmid pCL37 from which you have removed sites HindIII, XhoI and XmaJI. Next, the method of megaprimer [9, 10] in the DNA-fragment light_delta2 introduce recognition sites of endonucleases EcoRV and HindIII in the area corresponding to the ends of the VL-phase encoding variable domain light chain antibody. To do this, using PCR with primers 5 and 12 on the matrix light_delta2 get the DNA fragment, which after cleaning is complete by the matrix light_delta2 and amplificateur in the presence of oligonucleotides 5 and 6 with the formation of the fragment light_mod3. Using PCR with primers 6 and 11 to the matrix light_mod3 get the DNA fragment, which after cleaning is complete by the matrix light_mod3 and amplificateur in the presence of oligonucleotides 5 and 6 with the formation of the fragment light_mod4. This fragment join�tween with the original plasmid pCL37, hydrolyzed by sites NheI and ApaI, in a ligation reaction with the formation of plasmid pCLm4.

2. DNA fragment IGKC, previously obtained, is treated with the restriction endonucleases HindIII and BamHI and be ligated with a vector fragment obtained by hydrolysis of plasmid pCLm4 the same endonucleases. The result is a plasmid DNA pCLm4-IGKC.

3. Integration into the plasmid pCLm4 DNA fragment VL-14D5 encoding a variable domain of a murine monoclonal antibody 14D5. Fragment VL-14D5 previously obtained, amplificateur method pnrm. in the presence of primers 11 and 12. The obtained fragment was treated with restriction endonucleases EcoRV and HindIII and be ligated with a vector fragment obtained by hydrolysis of plasmid pCLm4-IGKC the same endonucleases. The result is a plasmid DNA pCLm4-14D5.

4. Cloning of the gene encoding the light chain of the chimeric antibody ch14D5, plasmid DNA pcDNA™3.1/Hygro(+). Plasmid DNA pCLm4-14D5 treated with endonucleases NheI and ApaI, resulting in a fragment size of 741 BP the fragment to be ligated with a vector DNA fragment formed during the hydrolysis of plasmid pcDNA™3.1/Hygro(+) the same endonucleases. The correctness of the embedding confirmed by sequencing using any available method, for example, using the kit BigDye® Terminator v3.1" (Applied Biosystems) and an automated capillary sequencer ABI 3730XL Geetic Analyser (Applied Biosystems). The result is a plasmid DNA pCLm4/hygro-14D5, a map of which is shown in Fig. 3.

Example 4. Construction of plasmids pCHm2-14D5 containing the gene encoding the heavy chain of the chimeric antibody ch14D5.

Construction of plasmids pCHm2-14D5 carried out in several stages:

1. In the vector plasmid RSN injected sites of the restriction endonucleases XhoI and Ass. For this PCR on the matrix rn with primers 5 and 14 have the DNA fragment sigVH. Next, by PCR using primers 13 and 14 based on the matrix sigVH get the DNA fragment, which after cleaning is complete and amplificateur in the presence of oligonucleotides 5 and 14 with the formation of the fragment sigVHmod. By PCR using as a template DNA the plasmid RCN in the presence of primers 5 and 15 receive the fragment h-pA. Then based on the matrix h-pA method of megaprimer in the presence of primers 5 and 15 complete fragment sigVHmod to fragment hm2a, which contains the gene for the heavy chain of the antibody introduced with the ends of the VH-phase sites of endonucleases XhoI and Ass. Fragment hm2a combine with the original plasmid RSN, hydrolyzed sites of the endonucleases NheI and XbaI, in a ligation reaction with the formation of plasmid pCHm2.

2. DNA fragment IGHG1, previously obtained, is treated with restriction endonucleases Acc65I and XbaI and be ligated with a vector fragment obtained by hydrolysis of plasmid pCHm2 these same Antonuk�the EEA. The result is a plasmid DNA pCHm2-IGHG1.

3. Fragment VH-14D5 previously obtained, amplificateur by PCR in the presence of primers 13 and 14. The obtained RTD-fragment treated with the restriction endonucleases XhoI and Acc65I and be ligated with a vector fragment obtained by hydrolysis of plasmid pCHm2-IGHG1 the same endonucleases. The correctness of the embedding confirmed by sequencing using any available method, for example, using the kit BigDye® Terminator v3.1" (Applied Biosystems) and an automated capillary sequencer ABI 3730XL Genetic Analyser (Applied Biosystems). The result is a plasmid DNA pCHm2-14D5, a map of which is shown in Fig. 4.

Example 5. Getting a full-sized chimeric antibody ch14D5 in eukaryotic mammalian cells.

Transient expression of a full-sized chimeric antibodies against tick-borne encephalitis virus is carried out as a result of co-transfection of Chinese hamster ovary cell line Cho-K1 derived expression plasmids pCLm4/hygro-14D5 and pCHm2-14D5 containing the genes for the light and heavy chains of the antibody. Before transfection the cells of the Cho-K1 cultured at 37°C in atmosphere of 5% CO2in DMEM (Gibco) containing 10% fetal bovine serum. Cells seeded on 6-well plates and grown to a density of 90-95%. The transfection is carried out using a reagent "Lipofectamine 2000" (Invitogen) according to the manufacturer's instructions. To do this, 4.5 ml of DMEM is added 36 µg DNA pCLm4/hygro-14D5 and 36 µg DNA pCHm2-14D5. At the same time mixed with 180 μl of reagent "Lipofectamine 2000" with 4.5 ml of DMEM. After a five minute incubation, both the obtained solution was combined and incubated for 20 min at room temperature. After incubation 0.5 ml of the suspension introduced into each well of the tablet containing cells Cho-K1. 4 hours after transfection the culture medium in the wells is replaced by 0.5 ml of medium "DMEM F12 advanced (Gibco) with 2% serum with reduced levels of immunoglobulins (Invitrogen). After 72 h, culture medium was collected and centrifuged 15 min at 15,000 g. The supernatant is separated, add sodium azide to a concentration of 0.05% and is used to isolate the antibody.

Example 6. The selection of recombinant antibodies using affinity chromatography.

Culture fluid in a volume of 500 ml is applied at 4°C at a rate of 0.5 ml/min on a column containing 5 ml of sorbent "Protein A Sepharose CL-4B (GE Healthcare) and balanced phosphate-saline buffer solution (PBS), which is a solution of 100 mm Nad, 50 mm Na2HPO4(pH of 7.4 at 25°C). The elution is carried out at 25°C and a speed of 1 ml/min using a chromatograph "BioLogic LP System (Bio-Rad). The column was washed with 25 ml of PBS, and then eluted immunoglobulins bull, present in the original serum, 25 ml of 0.1 M citrate buffer (pH 5.0 at 25°C). Next, 15 ml of 1 M citrate buffer (pH 3.0 at 25°C) eluted chimeric antibody in a test tube, containing 1.5 ml of a 1 M buffer Tris-HCl (pH of 8.0 at 25°C). The fraction containing the chimeric antibody was concentrated using concentrators "Amicon ultra-4" 30 kDa (Millipore) according to the manufacturer's instructions, at the same time replacing the buffer solution in PBS with 0.05% sodium azide. The concentration of antibody in the sample is determined spectrophotometrically at a wavelength of 280 nm, given that the solution of immunoglobulins IgG at a concentration of 1 mg/ml when the optical path length of 1 cm has an optical density of about 1.41 to [11]. The purified recombinant antibodies analyzed in regenerating and Sevostyanova fractionation conditions in 12% polyacrylamide gel with the addition of sodium dodecyl sulfate (Fig. 5).

Example 7. Determination of the affinity of the chimeric antibody ch14D5 to recombinantly similar to glycoprotein E of tick-borne encephalitis virus.

The kinetics of binding of the chimeric antibody ch14D5 with recombinant protein E [8] and the kinetics of dissociation of the complexes and thermodynamic affinity constant between these compounds was determined by surface plasmon resonance with the use of the device ProteOn XPR36 (Bio-Rad). To do this, activate the surface of one of the vertical channels of the biosensor chip GLC (Bio-Rad) using a set of "ProteOn Amine Coupling Kit (Bio-Rad) according to the instructions of the manufacturer. On the activated surface of immobile�comfort recombinant protein E at the rate of 250 µl of a solution with a concentration of 30 μg/ml protein in 10 mm acetate buffer (pH 4.5 at 25°C). Remaining activated groups block 1M solution of ethanolamine-HCl. As analyte using serial twofold dilutions of the chimeric antibody ch14D5 in PBS with the addition of 0.005% detergent tween-20, the maximum concentration of the antibody is 7.5 μg/ml. the Association is carried out for 600 s at a flow rate of 25 μl/min, followed by dissociation for 1800 seconds at the same speed. Based on the obtained sensograms calculate the kinetic binding constants of the components and dissociation of complexes, which are used to calculate the equilibrium dissociation constants, and affinity constants. The obtained experimental value of the constant of affinity of the chimeric antibody ch14D5 to protein E was 6.0 x 10-11M. (Fig. 6).

Example 8. Determination of the ability of the chimeric antibody ch14D5 to provide emergency prophylaxis mice from viral encephalitis.

To confirm the ability of the chimeric antibody ch14D5 to provide emergency prevention model animals from tick-borne viral encephalitis use mice of the Balb/C weighing 10 g. Each group contains 10 animals. To study the dose-dependent effect of the drug is a chimeric antibody ch14D5 administered in two doses 8 mg/kg of animal weight and 1 mg/kg of animal weight. As the comparison drugs using human immunoglobulin p�of otiv tick-borne encephalitis for intramuscular injection 10% with a titer of specific antibodies in hi 1:160 (NPO "Microgen", Tomsk). In order to control the lethality of the virus uses the infected group of mice, following infection with tick-borne encephalitis virus does not impose any chimeric antibody or drugs comparison. As a control group of intact mice.

For cooking use virus suspension brain of mice-suckers infected with tick-borne encephalitis virus, strain "Absettarov" stored at -30°C. After thawing, the suspension by serial ten-fold dilution is diluted in 107 times in 0.9% NaCl solution (pH 7,2-7,4 at 25°C) with addition of 2% serum of cattle. The mice intraperitoneally lead, 0.2 ml of the suspension in the working dilution. Real lethal dose of the drug was calculated according to the method of reed-MENA [12]. To do this, prepare serial tenfold dilutions of brain suspensions of the virus from 10-7up to 10-10and injected the mice intraperitoneally with 0.2 ml (6 goals at each dilution). Real dose is introduced virus in the experiment is 240 LDO.

Determination of the ability of the chimeric antibody to provide emergency prophylaxis mice from tick-borne viral encephalitis as follows. A day after virus infection, the mice intravenously in the tail vein once injected pre-prepared working solutions and�investigated antibodies in 0.9% sodium chloride solution (pH 7,2-7,4 at 25°C) at doses of 8 mg/kg of body weight (80 µg/mouse) and 1 mg/kg of body weight (10 μg/mouse). As the comparison drug intramuscularly administered drug of human immunoglobulin against tick-borne encephalitis for intramuscular injection at a dosage of 10 mg/kg of body weight (100 μg/mouse). Mice are observed for 21 days after Contracting the tick-borne encephalitis virus, counting mortality of animals. The results of the experiment show that a single administration of the chimeric antibody in a dosage of 8 mg/kg and 1 mg/kg to mice infected 240 LD50tick-borne encephalitis virus, has provided 100% survival of animals (mortality 0%). The results of the experiment shown in Fig. 7.

Thus, for the first time constructed a plasmid pCLm4/hygro-14D5 and pCHm2-14D5 containing artificial genes of the light and heavy chains of the full-sized chimeric antibodies are created on the basis of the variable fragment of light and heavy chains of murine monoclonal antibody 14D5, const genes of the immunoglobulin IgG1/Kappa human cytomegalovirus promoter and polyadenylation site of the gene of bovine growth hormone. Transient expression in eukaryotic cell lines the Cho-K1 leads to the biosynthesis of polypeptides with the properties of light and heavy chains of the chimeric antibody, snap antibody of the class IgG1/Kappa interacting specifically the surface glycoprotein E of tick-borne encephalitis virus. Cons�Anta affinity chimeric antibodies to recombinant glycoprotein E is about 6,0·10 -11M. Obtained chimeric antibody ch14D5 entered in a dosage of 1 mg per kg of animal weight, provides emergency preventing mice from tick-borne viral encephalitis, which makes possible the use of chimeric antibodies ch14D5 as the basis for the creation of drugs for the treatment of viral encephalitis.

SOURCES of INFORMATION

1. Ammosov A. D. Tick-borne encephalitis: Information guide / A. D. Ammosov. - Koltsov: Institute means honey. diagnosis of CJSC "Vector-best", 2006. - 115 p.

2. Pniewska N. And., Rudakov N. In. Efficacy of immunoglobulin preparations for post-exposure prophylaxis of tick-borne encephalitis in Russia (Review of half a century of experience) // Medical Parasitology and parasitic diseases. -2010. -N1. - S. 53-59

3. Morrison, S. L., Johnson, M. J., Herzenberg, L. A., V. T. Oi Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. // Proc Natl Acad Sci USA. - 1984. - V. 81(21). - P. 6851-6855.

4. Boulianne G. L., Hozumi N, Shulman MJ. Production of functional chimaeric mouse/human antibody. // Nature. - 1984. - V. 312(5995). - P. 643-646.

5. Tsekhanovskaya N. A., Matveev L. E., Rubin S. G., Karavanov A. S., Pressman, E. K. Epitope analysis of tick-bome encephalitis (TBE) complex viruses using monoclonal antibodies to envelope glycoprotein of TBE virus (persulcatus subtype). // Virus Res. - 1993. - V. 30(1). - P. 1-16.

6. Levanov L. N., Matveev L. E., Goncharova E. P., Lebedev L. R., Ryzhikov A. B., Yun I.e., Batanova T. A., Shvalov A. N., Baykov I. K., Shingarova L. N., Kirpichnikov M. P., Tikunova N. V. Chimeric antibodies against tick-borne encephalitis virus // Vaccine. - 2010. - V. 28. - P. 5265-5271.

7. Tikunova N. In. Shingareva L. N., Yoon T. E., and others. Recombinant plasmid DNA pcL37 that encodes the polypeptide with properties of light chain antibodies against the vaccinia virus recombinant plasmid DNA RSN that encodes the polypeptide with properties of the heavy chain of the indicated antibody, and their application. RF patent №2317330 C2, op.20.02.2008

8. ] N.And., Belavin P. A., Malygin E. G., M. Rukavishnikov Y. Recombinant plasmid DNA PGSDEI encoding protein E of tick-borne encephalitis virus, and Escherichia coli strain - producer of recombinant protein E of tick-borne encephalitis virus. Patent RF 2136754,1999

9. Giebel L. B., Spritz R. A. Site-directed mutagenesis using a double-stranded DNA fragment as a PCR primer. // Nucleic Acids Res. - 1990. - V. 18(16). -P. 4947.

10. Ke, S. H., Madison E. L. Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR method. // Nucleic Acids Res. - 1997. -V. 25(16). -P. 3371-3372.

11. Hale G. Isolation and purification of monoclonal antibodies from tissue culture supernatant // Monoclonal antibodies. A practical approach. Shepherd P and Dean C. (ed). Oxford University Press. 2000. P. 149-180.

12. Reed L. J., Muench, H. A simple method of estimating fifty percent endpoints. // The American Journal of Hygiene. - 1938. - V. 27. - P. 493-497.

1. Recombinant plasmid DNA pCLm4/hygro-14D5 that encodes the polypeptide with properties of light chain chimeric antibodies against tick-borne encephalitis virus, size 6232 BP and a molecular weight of 4.11 MDA, containing in accordance with the physical map shown in Fig.3:
- NheI/ApaI - vector fragment of plasmid pcDNA™3.1/Hygro(+) (Invitrogen) 5491 BP containing the promoter-enhanc�R cytomegalovirus, the polyadenylation site and the site of termination of transcription of the bovine growth hormone gene β-lactamase (bla) gene hygromycin phosphotransferase (hpt);
- NheI/ApaI - fragment size 741 BP, containing an artificial gene encoding a hybrid protein in which the variable domain light chain mouse monoclonal antibody 14D5 engaged in the protection against tick-borne encephalitis virus, coupled with the constant domain of the Kappa chain of human antibodies having the nucleotide sequence of SEQ ID NO: 1, shown in Fig. 1;
genetic markers: gene β-lactamase (bla), which determines the stability of the transformed plasmid pCLm4/hygro-14D5 cells of bacteria to ampicillin, a gene of hygromycin phosphotransferase (hpt), which determines the resistance to hygromycin In for breeding transfected with plasmid pCLm4/hygro-14D5 mammalian cells;
unique recognition sites of the restriction endonucleases, with the following coordinates: BglII-13, NheI-896, EcoRV-980, HindIII-1284, ApaI-1637.

2. Recombinant plasmid DNA pCHm2-14D5 that encodes the polypeptide with properties of chimeric heavy chain antibodies against tick-borne encephalitis virus, size 6765 BP and a molecular weight 4,47 MDA, including:
- NheI/XbaI vector fragment of plasmid pcDNA™3.1(+) (Invitrogen) size 5332 BP containing the promoter-enhancer of the cytomegalovirus CMV, is the site of polyadenylation and termination section �transcriptie gene of bovine growth hormone BGH, gene β-lactamase (bla) gene for resistance to neomycin (neo);
- NheI/XbaI - fragment size 1433 BP, containing an artificial gene encoding a hybrid protein in which the variable domain of the heavy chain of murine monoclonal antibody 14D5 engaged in the protection against tick-borne encephalitis virus, is connected to a constant CN1-CN2-CN3 domains of a human IgG1 having the nucleotide sequence of SEQ ID NO: 1, shown in Fig. 2;
genetic markers: gene β-lactamase (bla), which determines the stability of the transformed plasmid pCHm2-14D5 cells of bacteria to ampicillin, a gene of resistance to neomycin (neo) for selection of the transfected by plasmid pCHm2-14D5 mammalian cells;
unique recognition sites of the restriction endonucleases, with the following coordinates: BglII-14, NheI-897, XhoI-1001, Acc65I-1313, XbaI-2330.

3. Chimeric antibody capable of binding the protein E of tick-borne encephalitis virus and provide emergency prophylaxis of tick-borne encephalitis in mice, including polypeptides with the properties of light and heavy chains of the chimeric antibody of the class IgG1/Kappa, received a joint transfection of mammalian cells with recombinant plasmid DNA pCLm4/hygro-14D5 and pCHm2-14D5 described in claim 1 and formula 2.



 

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1 tbl

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