RU2534343C1 - Method of production of monoclonal antibodies to protein p30 of african swine fever virus using recombinant constructions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention consists in priming of mice with plasmid DNA encoding the gene of protein of interest to the researcher, which provides in vivo expression, processing, corresponding post-translational 3D conformation and induces an immune response similar to the serum to the infectious agent. Synergism of priming with DNA-plasmid and booster of effect of administration of recombinant protein in soluble form provides high avidity of CD4+ and CD8+ T-cells and, as a consequence, the affinity and avidity of monoclonal antibodies obtained. Application of DNA immunisation method and subsequent administration of a recombinant protein expands the range of methods of production of hybridomas producing specific mAb to different antigens. The method was tested in preparation of monoclonal antibodies to epitopes of phosphoprotein vp30 of African swine fever virus. The monoclonal antibodies thus obtained are capable of recognising antigenic determinants of phosphoprotein vp30 of African swine fever virus with cross immunoreactivity to the recombinant protein produced in E.coli clone PTT9/ASFVp30/2.

EFFECT: increased yield of hybridomas producing mAb to the desired antigen and reduced time of receiving donors of immune lymphocytes and lines of hybridomas producing specific mAb.

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Description

The invention relates to the field of veterinary virology and biotechnology, in particular to hybridoma technology, and relates to a method for producing monoclonal antibodies to VP30 of the African swine fever virus by fusion of mouse myeloma cells and lymphocytes immunized with the combined introduction of plasmid DNA with the insertion of the gene encoding p30 of the African plague virus pigs (ASF) and recombinant protein p30 mixed with adjuvant. The invention can be used to obtain monoclonal antibodies (MCAs) to specific proteins, individual epitopes, proteins with low immunogenic (antigenic) activity, as well as minor proteins of microorganisms.

African swine fever (ASF), a viral disease characterized by high contagiousness and mortality, is now widespread in the European territory of the Russian Federation. The most important epizootological feature (“insidiousness”) of African swine fever is an extremely rapid change in the course of infection among domestic pigs from acute with 100% mortality to chronic and asymptomatic carriage and unpredictable spread. One of the immunodominant diagnostically significant ASF virus proteins is phosphoprotein p30 (30 kDa), which is located in the inner membrane of infected cells [1], has antigenicity and is synthesized at all stages of the infection cycle, starting from 12-16 hours after infection, accumulating in the cytoplasm, which allows the detection of viral antigen in the first day after infection. After the release of viral particles into the extracellular space, most p30 also remains in the cells [2].

Immunization of plasmid DNA carrying the target protein gene induces the formation of specific antibodies and the activation of T-lymphocytes having receptors of both types of histocompatibility molecules (MHCs) necessary for the induction of a full-fledged humoral and cellular immune response. Mazumder S. et al. (2011) [3] when studying the induction and duration of the immune response against leishmaniasis in BALB / c mice with various combinations of DNA / DNA, DNA / protein and protein / protein showed the advantage of immunization with priming plasmid DNA and subsequent administration of the protein in soluble form.

The plasmid pTT9 / ASFVp30 of the E. coli BL21 (DE3) plysS cell strain [4] used for priming in immunization with mice contains the CBD gene, which encodes a cellulose-binding domain in the structure of the recombinant protein, by which the recombinant p30 split protein can bind with cellulose matrices. An increase in the level of the humoral and cellular immune response is carried out by binding to specific hydrocarbon receptors (lectins) expressed on antigen-presenting cells, thereby enhancing the antigen-specific T and B cell response [6].

The closest analogues of the invention are the production of MCAs for the main structural proteins of the ASF virus: vp73, vp30 [7], strain 2F2 of hybrid animal cells producing MCAs for Vp72, but the effectiveness of the interaction of these antibodies with compounds of the antigenic complex is not known [10]. The domestic literature describes the preparation of MCA for ASF polypeptides. [13, 14]

All of the above methods for producing MCA for ASF polypeptides used a viral antigen of varying degrees of purification and concentration. However, even with deep purification, impurity proteins of 14, 31, and 73 kDa are present in the preparation intended for immunization aimed at obtaining MCA for p30 protein [13].

Close by the principle of obtaining MCAs of a given specificity is the "Method for producing hybridomas producing monoclonal antibodies to recombinant proteins" [15].

The aim of the invention is to improve the method of producing hybridomas - producers of monoclonal antibodies of a given specificity.

The objective of the invention is to develop a method for producing stable highly productive lines of hybrid cells secreting MABs specific for ASF virus Vp30 and its recombinant analogue using the DNA immunization method and the introduction of the corresponding recombinant protein. For the subsequent construction of highly specific and standard domestic test systems for detecting Vp30 in ASF virus-infected biological fluids and animal tissues, in ticks, food products and food raw materials based on the obtained MCAs.

This goal is achieved by priming the recipient of plasmid DNA encoding the gene of the desired protein. The synthesis of the protein encoded in plasmid in vivo provides its further processing for presentation in combination with MHC antigens of classes I and II to immunocompetent cells. Subsequent administration of the appropriate recombinant protein provides the synthesis of specific antibodies to the introduced immunogen.

This method of delivering genetic material has several advantages over conventional protein immunization. The most important of these is the triggering of both pathways of the immune response. Unlike protein antigens, in vivo antigen synthesis ensures the correct folding and post-translational modification of the protein molecule. The risk of side effects associated with the introduction of foreign, ballast proteins, etc., is reduced. In addition, the DNA plasmid carrying the gene for the target protein causes a prolonged expression of the antigen and, accordingly, an immune response. The synergism of priming and the booster effect provide high avidity of CD4 + and CD8 + T cells and, as a result, the affinity and avidity of the obtained antibodies.

Technical result of the invention: the percentage of yield of hybridomas producing MCA to the desired antigen is increased, the time for obtaining donors of immune lymphocytes and hybridoma clones producing MCA to antigenic determinants of the target protein, in this case, ASF virus p30 protein, is reduced.

The invention consists in the following.

A BALB / c mouse is immunized with the plasmid preparation pTT9 / ASFVp30 / 2 and, subsequently, recombinant protein p30, cells of the mouse myeloma line Sp2 / 0-Ag14.1 are fused with lymphocytes of the immunized mouse. Hybridomas are obtained, supernatants of the obtained hybridomas are immunochemically tested, hybridoma clones producing monoclonal antibodies with specificity and affinity for ASF Vp30 epitopes are selected. This, in turn, makes it possible to study the cycle and characteristics of the reproduction of this protein and the virus as a whole, as well as to determine Vp30 in ASF virus infected biological fluids and animal tissues, in ticks, food products and food raw materials of animal origin.

The method of obtaining monoclonal antibodies to Vp30 ASF virus using recombinant constructs is as follows.

Mice are immunized using the plasmid DNA priming step, followed by the introduction of recombinant p30 protein with adjuvants. Hybridomas are obtained by 10: 1 fusion of splenocytes from BALB / c immune mice and mouse SP2 / 0-Ag14.1 mouse myeloma cells. in the presence of a 50% solution of PEG-4000 (Sigma) and the subsequent screening steps, three-fold cloning and stabilization of the obtained hybridoma lines.

The invention is illustrated, but not limited to the following examples.

Example 1. A specific immune response in mice immunized with recombinant ASF virus p30 constructs.

BALB / c mice, 4-6 week old, weighing 18-20 g, are immunized using the priming step — 50 μg of plasmid DNA is introduced intramuscularly carrying the insertion of the gene for the conserved region of the p30 viral protein with a molecular weight of 14 kDa (recombinant clone pTT9 / ASFVp30 / 2) followed by the introduction of 100 μg of recombinant p30 protein adsorbed on IT cellulose or with incomplete Freund's adjuvant according to the scheme shown in table 1. Table 1
BALB / c mice immunization schedule and titer specific antibodies Introduction No. Interval after the first immunization way volume The number of input protein (mcg) The composition of the administered drug Antibody titer Log ₂ * administered antigen one 0 PC* 0.5 cm ³ fifty pTT9 / ASFVp30 / 2 2 10 PC 0.5 cm ³ one hundred Rec p30 on pulp 3-4 _* 3 24 in / br * 0.5 cm ³ one hundred Recp30 + Freund's Incomplete Adjuvant 5-6 four 54 PC 0.5 cm ³ one hundred Rec p30 on pulp 6-7 5 3 days before hybridization all L* 0.1 cm ³ fifty Rec p30 9 Note: s / c - subcutaneous injection at 3-5 points, intraperitoneal administration - intraperitoneal administration, intravenous - intrasplenic administration of antigen, log ₂ * - two-fold dilutions of serum, starting at 1:50.

From the data presented in the table it can be seen that immunization of mice according to this scheme can be used to induce a specific immune response and, therefore, to obtain MCA for ASF virus p30 protein.

Example 2. Obtaining hybridomas producing monoclonal antibodies to the ASF virus p30 protein using recombinant constructs.

BALB / c mice were immunized as shown in Example 1. In the hybridization experiment, mice having a specific antibody titer in indirect TF ELISA of at least 6-7 log _{2 were used} . Hybridomas were obtained by fusion in the presence of a 50% solution of PEG-4000 (Sigma) splenocytes of BALB / c immune mice and the mouse myeloma line SP2 / 0-Ag14.1 tested for the absence of cell revertants. in a ratio of 10: 1. The resulting cells were seeded in 96-well culture plates onto a feeder peritoneal macrophage layer and cultured at 37 ° C. in an atmosphere containing 5% CO ₂ . Hybrid clone selection was performed on HAT and HT medium, followed by cultivation in DMEM (Sigma) medium with 15% FST, L-glutamine, sodium pyruvate, glucose, amphotericin-B.

Antibody products were screened by hybrid clones according to the standard method of indirect TF ELISA, latexagglutination reaction based on recombinant p30 protein, ELISA dot blot using recombinant p30 protein, native ASF virus culture antigen and immunochemical staining of CVC-1 cells and P-cell immunochemistry -66b infected with ASF virus.

Cloning of antibody-producing hybridomas was performed by the method of limiting dilutions according to the standard method in 96-well culture plates on a layer of feeder peritoneal macrophages starting from 7-10 days of cultivation.

In order to determine the stability of the obtained clone hybridomas and for long-term storage, the clones were cryopreserved on a medium consisting of 90% fetal calf serum and 10% dimethyl sulfoxide. Hybrid clones were stored in Dewar vessels with liquid nitrogen (minus 196 ° C).

24 hours before freezing, the hybridoma cells were passaged with a sieving ratio of 1: 2. Cells precipitated by centrifugation (1500 rpm, 10 min) were resuspended in a cryoprotective medium (90% FST, 10% DMSO) and poured into ampoules at a concentration of 3-5 mln / ml, 2 cm ³ . The mixture was held at 4 ° C for 24 hours, at -70 ° C for 24 hours, then transferred to liquid nitrogen (minus 196 ° C).

Rapid thawing of the cells was carried out at 37-39 ° C. Hybridoma cells were diluted 10 times with growth medium without serum of cow embryos, precipitated by centrifugation at 1500 rpm for 10 minutes, the pellet was resuspended in growth medium containing 20% fetal serum of cows to a concentration of 3-4 × 10 ⁵ cells / cm ³ . The viability of the restored cells was determined by differential staining of the cells using a 0.4% trypan blue solution, pH 7.2-7.3, which was 70-80%.

Obtaining ascitic fluids containing MCA. 14 days before the introduction of hybridoma cells, BALB / c mice were pretreated with 2,6,10,14-tetramethylpentadecane (Pristane, Sigma, USA) at a dose of 0.3-0.5 cm ³ / animal. Next, the hybridized cells of one clone at a dose of (5-10) · 10 ⁶ cells were introduced into the abdominal cavity of the primed animals. Ascitic fluids containing MCA were obtained 7-15 days after administration.

Characteristics of produced MCAs:

- the target is the main conformational epitope of the structural protein vp30 ASF virus;

- specificity of binding to the conformational epitope of the vp30 ASF virus - 100% in TF ELISA;

- cross-immunoreactivity to the conformational epitope of a recombinant protein taken for immunization - 100%;

- isotype of produced immunoglobulins - IgG1 (ELISA, test system "Mouse-Hybridoma-Subtyping Kit" ("Sigma", USA).

Example 3. The use of MCA to p30 ASF virus obtained by immunization with genetic engineering constructs for the detection of ASF virus antigens by indirect immunofluorescence (RNIF).

For the formulation of RNIF, a standard technique was used. A goat IgG-goat IgG conjugate to mouse IgG in working dilution was used as an anti-species conjugate.

The result of the reaction was taken into account by the level of specific glow in reflected blue light on a luminescent microscope at a magnification of 1 × 90. MCA for recombinant p30 clones 1C2, 7F7, 2B7 and 5D11 specifically interacted only with ASF virus antigens of infected culture test preparations, causing characteristic specific granular fluorescence in the cytoplasm of infected cells. They did not interact with antigens of test preparations of the intact CV-1 cell culture and preparations of Teshen disease virus, classical swine fever and Aujeszky's disease - the absence of such a glow. A slight background of diffuse staining is possible. The activity of MkAt in RNIF was 1: 256-1: 512.

Literature

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2. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization / P. Gomez-Puertas, F. Rodriguez, J.M. Oviedo [et al.] // J. Virol. - 1996 .-- Vol. 70. - P.5689-5694.

3. Mazumder S. Potency, Efficacy and Durability of DNA / DNA, DNA / Protein and Protein / Protein Based Vaccination Using gp63 Against Leishmania donovani in BALB / c Mice [text] / S. Mazumder, M. Maji, A. Das, N. Ali // PLoS ONE - 2011 .-- No. 6 (2): e14644.

4. Pat. 2463343 RU 2463343 C1. The E. coli BL21 (DE3) pLysS cell strain, clone pTT9 / ASFVp30, containing a recombinant plasmid with an insertion of a region of the African swine fever virus CP204L gene encoding the p30 protein conformational epitope suitable for the manufacture of diagnostic preparations / B.O. Kopytov, S.Zh. Tsybanov, A.S. Kazakova, T.E. Yuzhuk, S.A. Belyanin, A.G. Guzalova, N.N. Vlasova, D.V. Sausages (RU). - No. 2011141517/10; announced on 10/13/2011; publ. 10/10/2012, Bull. No. 28. - 8 p.

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8. High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potencial use as diagnosis reagents / J.M. Oviedo, F. Rodriguez, P. Gomez-Puertas [et al.] // J. Virol. Methods - 1997 .-- Vol.64. - P.27-35.

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14. Mitin N.I., Sidorov S.I., Reutova E.G.

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Claims (1)

A method of producing hybridomas producing monoclonal antibodies to the structural protein p30 of the virus of African swine fever virus, comprising immunizing mice and obtaining hybrid clones, characterized in that plasmid DNA carrying the target protein gene is used to prime the recipient's immune system, followed by enhancing the immune response by introducing the appropriate recombinant protein .

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