阅读说明：本技术 非洲猪瘟预防和/或治疗性中和抗体、其制备方法与应用 (African swine fever preventing and/or treating neutralizing antibody, preparation method and application thereof ) 是由 曹文龙 孔迪 孙祥明 滕小锘 张大鹤 易小萍 于 2019-11-22 设计创作，主要内容包括：本发明提供了一种非洲猪瘟预防和/或治疗性中和抗体,其为嵌合单克隆抗体,包含重链和轻链,其中的轻链可变区具有SEQ ID No：6所示序列,重链可变区具有SEQ ID No：2所示序列,轻链恒定区、重链恒定区均来源于猪源抗体。本发明还提供了编码嵌合单克隆抗体的基因、包含所述编码基因的表达载体或转化体等。所述表达载体可以是将所述编码基因插入pAAV-CAG载体而得。本发明还提供了一种利用所述表达载体制备AAV的方法。本发明提供的所述抗体序列或转化体等可以用于制备非洲猪瘟病毒的检测试剂、用于在受试动物中诱导针对非洲猪瘟病毒抗原的免疫反应的药剂或者用于预防动物受非洲猪瘟病毒感染的药剂,提供迅速而持续长久的抗体保护,例如非洲猪瘟病毒疫苗。(The invention provides an African swine fever preventing and/or treating neutralizing antibody, which is a chimeric monoclonal antibody and comprises a heavy chain and a light chain, wherein the variable region of the light chain has the amino acid sequence shown in SEQ ID No:6 and the heavy chain variable region has a sequence shown in SEQ ID No:2, the light chain constant region and the heavy chain constant region are both derived from porcine antibodies. The invention also provides a gene encoding the chimeric monoclonal antibody, an expression vector or a transformant containing the encoding gene, and the like. The expression vector can be obtained by inserting the coding gene into pAAV-CAG vector. The invention also provides a method for preparing AAV by using the expression vector. The antibody sequence or the transformant and the like provided by the invention can be used for preparing a detection reagent of the African swine fever virus, a medicament for inducing an immune response to an African swine fever virus antigen in a tested animal or a medicament for preventing the animal from being infected by the African swine fever virus, and provides rapid and long-lasting antibody protection, such as an African swine fever virus vaccine.)

1.A chimeric monoclonal antibody comprising a heavy chain and a light chain, characterized in that: the light chain comprises a light chain variable region and a light chain constant region, and the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the light chain variable region has the sequence shown in SEQ ID No:6 or a conservative variant thereof, and the heavy chain variable region has a sequence shown in SEQ ID No:2 or a conservative variant sequence thereof; the light chain constant region and the heavy chain constant region are both derived from porcine antibodies.

2. The chimeric monoclonal antibody according to claim 1, characterized in that: the light chain constant region has the sequence of SEQ ID NO: 8 or a conservative variant thereof, and the heavy chain constant region has a sequence shown in SEQ ID No: 4 or conservative variant sequence thereof.

3. The chimeric cloned gene sequence according to claim 1, characterized in that: the heavy and light chains are bound by interchain disulfide bonds.

4. The chimeric monoclonal antibody according to claim 1, characterized in that: the heavy chain and the light chain of the chimeric monoclonal antibody have the amino acid sequences shown in SEQ ID No: 14. SEQ ID No: 16 or a conservative variant thereof.

5. A gene encoding the chimeric monoclonal antibody of any one of claims 1-4, wherein the gene encoding the light chain has the amino acid sequence of SEQ ID No:15 or a conservative variant sequence thereof, and the gene encoding the heavy chain has the sequence shown in SEQ ID No:13 or a conservative variant thereof.

6. An expression vector or a transformant containing the gene encoding the chimeric monoclonal antibody according to claim 5.

7. The expression vector or transformant according to claim 6, characterized in that: the expression vector is obtained by inserting the gene coding the chimeric monoclonal antibody between EcoRI and Hind III of a pAAV-CAG vector, and the transformant is obtained by cotransfecting the expression vector, a pHelper vector and a pAAV-RC vector into a host cell; preferably, the host cell comprises a HEK293 cell.

8.The expression vector or transformant according to claim 6, characterized in that: the gene coding the heavy chain and the gene coding the light chain are inserted behind the CAG promoter in the expression vector, and the gene coding the heavy chain and the gene coding the light chain are connected through a connecting peptide; preferably, the linker peptide comprises an FMDV 2A peptide.

9. Use of the chimeric monoclonal antibody according to any one of claims 1 to 4, the gene encoding the chimeric monoclonal antibody according to claim 5, or the expression vector or transformant according to any one of claims 6 to 8 for the preparation of a test reagent for African swine fever virus, a medicament for inducing an immune response against an antigen of African swine fever virus in a test animal, or a medicament for preventing infection of an animal with African swine fever virus.

10. A method for producing a recombinant AAV, comprising: culturing host cells, adding a transfection reagent when the cells grow to 50% -70% fusion, adding the expression vector of any one of claims 6-8, the pAAV2-RC vector and the pHelper vector to co-transfect the host cells, culturing at room temperature for more than 5h, then changing a fresh culture medium, continuing culturing and collecting the cells, repeatedly freezing and thawing the collected cells for lysis, and then extracting the recombinant AAV through post-treatment; preferably, the host cell is a HEK293 cell; preferably, the transfection reagent is polyethyleneimine; preferably, the expression vector, the pAAV2-RC vector and the pHelper vector are added in an equal mass ratio.

Technical Field

The invention relates to a neutralizing antibody, in particular to an African swine fever preventing and/or treating neutralizing antibody, a preparation method and application thereof, and belongs to the technical field of animal immune drugs.

Background

African Swine Fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African Swine Fever Virus (ASFV), and is clinically manifested as high fever, cutaneous congestion, edema, generalized bleeding of organs, and changes in respiratory and nervous system functions. The disease has short morbidity process, a latent period of 5-15 days, high morbidity and mortality, and even the mortality can reach 100%. In countries or regions infected by this pathogen, huge economic losses can be caused, food safety is seriously threatened and the pig industry is affected. The world animal health Organization (OIE) classifies the disease as a type A epidemic disease, and the disease is specified as an animal disease in China.

The ASFV is an icosahedral symmetrical structure with a diameter of 175-215 nm. The virus nucleic acid is double-stranded DNA, has the length of 170 kb-190 kb, contains 151 open reading frames, and can encode 150-200 proteins, wherein the main structural proteins of virus particles comprise P72, P54, P220, P62, CD2V and the like. The P54 protein is used as a main structural protein of the African swine fever virus, is a main binding site of a serum antibody, has good immunogenicity, and can be used as a marker for early detection of the African swine fever virus.

The resistance of the African swine fever virus in tissues and environment is strong, no effective treatment measures exist at present, the most effective method in the whole world is to kill all pigs in an epidemic area and nearby areas, and disastrous results are brought to pig raising enterprises and pig farmers.

Moreover, there is currently no vaccine that is particularly effective against african swine fever. Vaccines prepared by traditional methods, such as purified and inactivated virus, formaldehyde inactivated virus-infected porcine alveolar macrophages, and porcine infected peripheral blood leukocyte supernatants do not induce protective immunity. That is, cellular immunity and humoral immunity are limited in preventing and treating infection of African swine fever and in eliminating viruses in general.

Early results of the study tended to suggest that African swine fever virus could not induce the production of neutralizing antibodies (HessWR.1981.African swine fever mover: a reassessment. Advances in veterinary science and comparative media 25:39-69) because the detection of neutralizing antibodies using the plaque reduction method was difficult, and more particularly, because the formation of macroscopic plaques was very difficult with some isolated ASFVs, especially ASFVs with a low number of generations. However, with the advancement of research methods, the detection of viruses has been facilitated by introducing marker genes into viruses, and more studies have been carried out on neutralization of serum or monoclonal antibodies of some isolated ASFVs recovered from infection with viruses (Borca MV, Irusta P, Carrillo C, Afanso CL, Burrage T, RockDL.1994.African switch virus structural protein P72 antibodies for neutral epitope 201: 413-418; Gomez-Puertas P, Rodriguez F, Oedio, Ramio-Ibannez F, Ruiz-Gonzaro F, Alonso C, Escribane D. 1996.Neutralizing virus to viral infection of viral infection 19, vitamin K J. J.R. F5632. and F, vitamin K J.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R, carnero ME, Caballero C, Martinez J.1986.inhibition of African Swinhaeffector infection in the presence of immune sera in vivo and in vitro. am J VetRs 47: 1249-1252; ruiz Gonzalvo F, Cabillaro C, Martinez J, CarneroME.1986.Neutralization of African swine farm by sera from African swine farm by-resistant pigs. am J Vet Res 47: 1858-1862). There are at least two mechanisms by which sera from pigs immunized with live attenuated vaccines neutralize viral infection of Vero or porcine alveolar macrophages. One mechanism is to prevent binding of the virus to the cell, and the other mechanism is to prevent endocytosis of the virus (Gomez-Puertas P, Rodriguez F, Ovido JM, Ramiro-Ibanez F, Ruiz-Gonzalo F, Alonso C, Escribano JM.1996.neutral antibodies to differential proteins of African swineverture virus in vivo virus tissue attachment and interaction. journal of virology70: 5689-.

Studies of sera from pigs infected with virus that are recovering health have shown that the P72, P30, and P54 proteins are the three most immunogenic outer membrane proteins in ASFV (Afanso CL, Alcaraz C, Brun A, Sussman MD, Onisk DV, Escribano JM, Rock DL.1992. Characterisation of P30, a high antibody membrane and secreted protein of African swine farm virus, virology 189: 373). Antibodies against P72 and P54 proteins neutralize the virus only before the virus adsorbs susceptible cells, while anti P30 antibodies neutralize the virus before or after the virus adsorbs susceptible cells. Therefore, it is considered that the antibodies against P72 and P54 inhibit the first step of the viral replication cycle, i.e., the viral adsorption process. While antibodies against the P30 protein inhibit the endocytosis of the virus (Gomez-Puertas P, Rodriguez F, Ovido JM, Ramiro-Ibanez F, Ruiz-Gonzalo F, Alonso C, Escribano JM.1996. neutralling antibodies to differential proteins of African swinevirus with bit virus attachment and interaction. journal of virology70: 5689-.

Treatment of ASFV virus with detergent releases the P30 and P54 proteins, which are able to adsorb to the surface of ASFV-sensitive porcine alveolar macrophages, and this adsorption can be inhibited by the corresponding antibodies. In addition, the insect cells are used for expressing ASFV hemagglutinin protein CD2V, a protein homologous with CD2 protein, and the immune pig can generate temporary hemagglutination inhibition antibody.

The porcine immune attenuated strain OUR/T88/3 was able to defend against challenge by the homologous virulent strain OUR/T88/1, but when CD8+ lymphocytes in vivo were depleted, it was not able to completely defend against challenge by the virulent strain, suggesting that cellular immunity of CD8+ lymphocytes plays a critical role in immune protection, whereas neutralizing antibodies alone are not able to adequately defend against virus challenge (Gomez-Puertas P, Rodriguez F, Ovido JM, Ramio-Ibanez F, Ruiz-Gonzalo F, Alonso C, Esacro JM.1996.neutral antibodies, colloidal protein of animal virus in vivo virus infection bone virus infection, Golvo virus infection, Golvin J12547, Marlvin J12547, CaballeroC, Martinez J, Carnero ME.1986.Neutralization of African swing boiler virus from African swing boiler-resistant pills. am J Vet Res 47: 1858-; barderas MG, Rodriguez F, Gomez-Puertas P, Aviles M, Beitia F, Alonso C, EscribanoJM.2001.Antigenic and immunogenic properties of a chicken of twosimulune bacterial viruses in Arch Virol 146: 1681-; Gomez-Puertas P, Rodriguez F, Ovido JM, Brun A, Alonso C, Escribano JM.1998, the African swine boiler virus proteins P54 and P30 area involved in two variants of virus approach and both constraint to the antibiotic-modified antibody response, virology 243: 461-; onisk DV, Borca MV, Kutish G, Kramer E, Irusta P, Rock DL.1994.Passively transferred African swine revolute antibodies protective swine against viral infection. virology 198: 350-.

On the other hand, one to weak low virulent strain that has been adapted to culture in CV1 cells was injected into pigs, and antibodies isolated from serum of recovered pigs immunized against otherwise healthy pigs that were able to defend against challenge with the virulent strain. Antibodies isolated from recovered porcine serum were able to neutralize wild strains including E75, E70, Lisbon60, etc., reducing their ability to infect Vero cells or macrophages by 86-97%.

Some studies have shown that 85% of pigs vaccinated with anti-ASFV antibodies are able to withstand challenge with the virulent strain E75 strain, while the remainder of pigs vaccinated with negative serum alone or PBS alone are 100% dead. The clinical manifestations of the vaccinated pigs were the same as those of normal pigs except for delayed and transient febrile reactions, whereas the control group showed significant clinical signs of ASFV 4 days after challenge. In addition, the virus content in the blood of the pigs inoculated with the antibody group is reduced by more than 10000 times, which indicates that the anti-ASFV antibody alone can protect the attack of ASFV virulent strains. There is also data indicating that antibody-mediated protection can delay disease progression early. Other studies have shown that some protection is obtained in piglets when they pass colostrum for antibodies against ASFV.

Research and development of a drug effective in preventing and treating African swine fever is the most serious challenge and urgent task for the global medical science community.

There are also some studies on African swine fever virus vaccines reported at present. For example, CN110093324A and CN106459931A propose attenuated strains of african swine fever virus with different gene knock-outs as live attenuated african swine fever vaccines, although the protective effect of the vaccines is good under laboratory conditions, the live attenuated vaccines are used with very large side effects, and because the research on the african swine fever virus is not thorough, the genes and mechanisms related to the virus virulence are not well understood, and the live attenuated vaccines have not been subjected to rigorous and comprehensive safety assessment, the biological risk as vaccines is extremely high. CN109836478A, CN104311660A and the like propose a monoclonal antibody against ASFV P11.5 and VP73 proteins. The monoclonal antibodies proposed in this patent are only useful as diagnostic reagents, and the antibodies have no neutralizing ability and cannot be used in therapeutic as well as prophylactic vaccines. CN109734810A patent application discloses a method for preparing anti-african swine fever virus and CD dual-target swine humanized antibody. This patent provides a method for preparing bispecific antibodies without providing antibody sequences, which uses recombinant CHO cells to express the antibody, which is then purified and injected into swine, but repeated injections are required at short intervals because the antibody half-life is very short and does not provide continuous protection. The antibody sequence and protection mechanism in the preparation method of the antibody are completely different from the antibody sequence and protection mechanism. The method utilizes the injection of recombinant AAV to continuously produce neutralizing antibodies for the treatment and prevention of ASFV.

Disclosure of Invention

The invention mainly aims to provide an African swine fever preventing and/or treating neutralizing antibody, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a chimeric monoclonal antibody, which comprises a heavy chain and a light chain, wherein the light chain comprises a light chain variable region and a light chain constant region, and the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the light chain variable region has the sequence shown in SEQ ID No:6 or a conservative variant thereof, and the heavy chain variable region has a sequence shown in SEQ ID No:2 or a conservative variant sequence thereof; the light chain constant region and the heavy chain constant region are both derived from porcine antibodies.

In some embodiments, the light chain and the heavy chain of the chimeric monoclonal antibody have the amino acid sequences of SEQ ID nos: 14. SEQ ID No: 16 or a conservative variant thereof.

The embodiment of the invention also provides a gene for coding the chimeric monoclonal antibody, wherein the gene for coding the light chain has the nucleotide sequence shown in SEQ ID No:15 or a conservative variant sequence thereof, and the gene encoding the heavy chain has the sequence shown in SEQ ID No:13 or a conservative variant thereof.

The embodiment of the invention also provides an expression vector or a transformant containing the gene for encoding the chimeric monoclonal antibody.

In some embodiments, the expression vector is obtained by inserting the gene encoding the chimeric monoclonal antibody between the cleavage sites EcoRI and HindIII of the pAAV-CAG vector, and the transformant is obtained by co-transfecting a host cell with the expression vector, a pHelper vector, and a pAAV-RC vector.

The embodiment of the invention also provides application of the chimeric monoclonal antibody, the gene for coding the chimeric monoclonal antibody or the expression vector or the transformant in preparing a detection reagent of the African swine fever virus, a medicament for inducing an immune response to an African swine fever virus antigen in a tested animal or a medicament for preventing the animal from being infected by the African swine fever virus.

The embodiment of the invention also provides a preparation method of the virus particles, which comprises the following steps: culturing host cells, adding a transfection reagent when the cells grow to be 70% fused, adding the expression vector, the pAAV2-RC vector and the pHelper vector to co-transfect the host cells, culturing at 37 ℃ for 5 hours, then changing a fresh culture medium, continuing culturing and collecting the cells, repeatedly freezing and thawing the collected cells for cracking, and then extracting the virus particles by post-treatment.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following outstanding advantages and effects:

(1) for example, 293 cells and other eukaryotic cells express ASFV P54 protein antigen, and the protein is expressed by soluble secretion, correct in folding and strong in immunogenicity;

(2) the selected monoclonal antibody has high neutralizing potency, and can effectively resist the adsorption infection of the virus to cells.

(3) The gene sequence of the antibody is obtained by extracting the total mRNA of the hybridoma cell and an RT-PCR amplification method, and the light chain and the heavy chain variable region of the monoclonal antibody are hybridized with the light chain and the heavy chain constant region of the pig IgG antibody to form the chimeric antibody, so that the high neutralizing activity of the antibody is kept, the immunogenicity of the antibody is reduced, and the effect of the antibody is improved.

(4) The AAV is used as a gene vector to transduce the antibody gene into the pig cell, so that the advantages of high safety, small virus particles, weak antigenicity and the like of the AAV vector can be fully exerted, the pig can quickly and continuously secrete the monoclonal antibody for a long time, and the pig can be protected for a long time. In the existing method of using monoclonal antibody injection, because the half-life of the antibody is very short, in order to achieve immunoprophylaxis, repeated injection is needed at short intervals, which is costly and complicated.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic representation of the pCI-P54His plasmid in example 1;

FIG. 2 is an SDS-PAGE gel electrophoresis chart in example 2;

FIG. 3 is a schematic diagram of the pAAV-CAG-Cap vector in example 6.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

One aspect of the embodiments provides a chimeric monoclonal antibody comprising a heavy chain and a light chain, the light chain comprising a light chain variable region and a light chain constant region, and the heavy chain comprising a heavy chain variable region and a heavy chain constant region; the light chain variable region has the sequence shown in SEQ ID No:6 or a conservative variant thereof, and the heavy chain variable region has a sequence shown in SEQ ID No:2 or a conservative variant sequence thereof; the light chain constant region and the heavy chain constant region are both derived from porcine antibodies.

In some embodiments, the light chain constant region has the amino acid sequence of SEQ ID No: 8 or a conservative variant sequence thereof.

In some embodiments, the heavy chain constant region has the amino acid sequence of SEQ ID No: 4 or a conservative variant sequence thereof.

In some embodiments, the heavy and light chains are linked by interchain disulfide bonds.

In some embodiments, the amino acid sequence of the chimeric monoclonal antibody is as set forth in SEQ ID No: 12 or a conservative variant sequence thereof.

In some embodiments, the light chain variable region and the light chain constant region comprise a light chain and the heavy chain variable region and the heavy chain constant region comprise a heavy chain, and the gene encoding the light chain has the amino acid sequence of SEQ ID No:15 or a conservative variant sequence thereof, and the coding gene of the heavy chain has a sequence shown in SEQ ID No:13 or a conservative variant thereof.

In some embodiments, the gene encoding the light chain variable region has the amino acid sequence of SEQ ID No:5 or a conservative variant sequence thereof.

In some embodiments, the gene encoding the heavy chain variable region has the amino acid sequence of SEQ ID No:1 or a conservative variant sequence thereof.

In some embodiments, the gene encoding the light chain constant region has the amino acid sequence of SEQ ID No: 7 or a conservative variant sequence thereof.

In some embodiments, the gene encoding the heavy chain constant region has the amino acid sequence of SEQ ID No: 3 or a conservative variant sequence thereof.

In some embodiments, the gene encoding the chimeric monoclonal antibody has the amino acid sequence of SEQ ID No:11 or a conservative variant thereof.

In the present specification, the aforementioned conservative variant sequence refers to a nucleotide and/or amino acid sequence formed by substitution, insertion or deletion of a nucleotide or amino acid at one or more sites in the original nucleotide sequence and/or the original amino acid sequence. Preferably, the conservative variant sequence is at least 95% identical to the original sequence.

In another aspect of the embodiments of the present invention, there is provided an expression vector or a transformant containing the gene encoding the chimeric monoclonal antibody.

In some embodiments, the expression vector (which may be defined as an AAV expression vector) is the gene encoding the chimeric monoclonal antibody inserted between EcoRI and HindIII of the pAAV-CAG vector.

In the present specification, the pAAV-CAG vector is prepared by a method which is conventional in the art, or is commercially available. Wherein the restriction sites of the multicloning site of the pAAV-CAG vector are those conventional in the art as long as the gene encoding the chimeric monoclonal antibody can be cloned into the pAAV-CAG vector, and the restriction sites are preferably those of restriction enzymes EcoRI and HindIII.

In some embodiments, the sequence of the gene encoding the chimeric monoclonal antibody is preferably a homologue of SEQ ID No. 11, which may be a promoter variant. The promoter or signal sequence preceding the nucleic acid sequence may be altered by one or more nucleic acid substitutions, insertions or deletions without these alterations having a negative effect on the function of the promoter. Furthermore, the expression level of the target protein can be increased by changing the sequence of the promoter or even completely replacing it with a more efficient promoter from a different species of organism. Homologs of SEQ ID No. 11 also include a class of polynucleic acids having a base sequence capable of hybridizing with a polynucleic acid having a sequence shown in SEQ ID No. 11 under standard conditions in a manner described in Current Protocols in Molecular Biology, described in Molecular cloning. The homologue of SEQ ID No. 11 is preferably one in which a signal peptide molecule is added before the sequence of SEQ ID No. 11, which serves to control functions such as transfer and localization of the sequence of SEQ ID No. 11 in cells, wherein the signal peptide molecule is a signal peptide molecule that is conventional in the art, and the sequence of the signal peptide molecule can be obtained by artificially synthesizing a polypeptide sequence or by molecular cloning techniques, in a manner described in the general protocol in molecular biology, described in molecular cloning (Cold Spring Harbor Laboratory Press).

In some embodiments, the gene encoding the heavy chain and the gene encoding the light chain are inserted after the CAG promoter in the expression vector, and the gene encoding the heavy chain and the gene encoding the light chain are connected by a connecting peptide. Preferably, the linking peptide includes, but is not limited to, the FMDV (foot and Mouth Disease Virus)2A peptide.

In the present specification, the transformant is prepared by transforming the expression vector of the present invention into a corresponding host cell. The host cell is conventional in the art, preferably a eukaryotic cell, more preferably a HEK293 cell, as long as it is sufficient that the AAV vector is stably self-replicating. The HEK293 cell line is prepared by a conventional method in the art or is commercially available.

In some embodiments, the transformants are obtained by co-transfecting the expression vector (AAV expression vector described above) with a pHelper vector, a pAAV-RC vector, and a host cell.

Another aspect of the embodiments of the present invention provides a method for preparing a virus particle, including: culturing host cells, adding a transfection reagent when the cells grow to be 70% fused, adding the expression vector, the pAAV2-RC vector and the pHelper vector to co-transfect the host cells, culturing at 37 ℃ for 5 hours, then changing a fresh culture medium, continuing culturing and collecting the cells, repeatedly freezing and thawing the collected cells for cracking, and then extracting the virus particles by post-treatment.

In some embodiments, the host cell is a host cell conventional in the art, preferably a eukaryotic cell, more preferably a HEK293 cell.

In some embodiments, the transfection reagent is a transfection reagent conventional in the art, provided that it is sufficient to transfect the foreign plasmid into the host cell, preferably a polyethyleneimine reagent (PEI reagent). The preparation method of the polyethyleneimine reagent is a conventional preparation method in the field, or is commercially available.

In some embodiments, the AAV vector, pAAV2-RC vector, and pHelper vector are preferably added in equal mass ratios.

In some embodiments, the three plasmids are preferably added in an amount of 6-16. mu.g.

When the addition amount of the three plasmids is not within the above range, the AAV virus packaging efficiency is low, and even the AAV virus cannot be packaged.

In the specification, the pAAV2-RC vector and the pHelper vector are pAAV2-RC vector and pHelper vector which are conventional in the field, and the preparation methods of the vectors are conventional in the field or are commercially available.

In some embodiments, the method for preparing the virus particles may further comprise a concentration step, and the concentration method is a conventional concentration method in the field, for example, the collected eluate containing the virus may be concentrated and processed by a concentration column. Wherein the concentration column is a concentration column conventional in the art as long as it can concentrate the resulting virus particles. The preparation method of the concentration column is a conventional preparation method in the field, or is commercially available.

Another aspect of the embodiments of the present invention provides an AAV viral particle comprising the expression vector. The AAV viral particles can be prepared by the methods described previously.

In another aspect of the embodiments of the present invention, there is provided a use of the chimeric monoclonal antibody, the gene encoding the chimeric monoclonal antibody, the expression vector or transformant, or the AAV viral particle in the preparation of a detection reagent for african swine fever virus, a medicament for inducing an immune response against an african swine fever virus antigen in a test animal, or a medicament for preventing an infection of an animal with african swine fever virus.

For example, some embodiments of the invention provide for the use of said expression vector or said AAV viral particle in the preparation of an african swine fever virus vaccine.

Another aspect of the embodiments of the present invention provides an immunological composition comprising the expression vector as described above as an active ingredient and at least one pharmaceutically acceptable carrier. The pharmaceutical carrier of the present invention is a conventional pharmaceutical carrier in the art, preferably a filler, diluent or excipient, etc., and is not limited thereto. Further, the immune composition may be prepared in various dosage forms including, but not limited to, tablets, capsules, powders, pills, granules, syrups, solutions, suspensions, emulsions, suspensions, injections, or powder injections; preferably, the dosage form is injection, such as intravenous injection, intraperitoneal injection and the like.

In some more specific embodiments of the present invention, mice are immunized with 293 cell expressed ASFV P54 protein as an antigen to prepare hybridoma cells, cell lines capable of secreting monoclonal antibodies with high neutralizing titers are selected, total RNA of the hybridoma cells is extracted, the gene sequences of the monoclonal antibodies are amplified by reverse transcription, and the variable region sequences of the heavy and light chains of the monoclonal antibodies and the published constant region sequences of the heavy and light chains of the porcine antibodies are made into a humanized chimeric antibody. The chimeric antibody gene is then codon optimized and cloned into an AAV vector, which can be used for the treatment or prevention of african swine fever virus, e.g. for the preparation of an african swine fever virus vaccine.

It is to be understood that the definitions of certain terms used herein are known to those of ordinary skill in the art unless otherwise indicated. For example:

an antibody consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain is flanked by a variable region (V) followed by a number of constant regions (C). Each light chain is flanked by a variable region (V) followed by a number of constant regions (C).

The variable regions of the natural heavy and light chains each contain four FR regions, most of which adopt an β -fold configuration, linked by three CDRs, three CDR links which form a loop link and in some cases a portion of β fold.

Adeno-associated virus (AAV), which belongs to the genus dependovirus of the family parvoviridae, is a DNA replication-deficient virus, and replication and propagation of AAV virus need to depend on the participation of other helper viruses such as adenovirus. The genome of an AAV virus is a single-stranded DNA, about 4.7Kb in length, and is composed of three important elements, namely: a pair of Inverted Terminal Repeats (ITRs), a Rep region and a Cap region. ITRs can form T-type secondary structures and are the only cis-acting elements required for AAV virus replication, packaging, integration and rescue (molecular structure of adeno-associated virus variant DNA. the Journal of biological chemistry 1980, 255: 3194. sup. 3203). Systems have been developed in the present stage that do not require helper viruses to accomplish recombinant adeno-associated virus packaging (high pure viral infection-associated virus vectors active and free of detectable salts and wild-type viruses HumGene Heat 1999,10: 1031-. The packaging system comprises three plasmids and a cell line, wherein the three plasmids are respectively pAAV-MCS carrying ITRs elements, eukaryotic promoters and exogenous gene insertion sites; pAAV-RC providing the Rep region and Cap region necessary for AAV viral packaging; pHelper derived from helper proteins E2A, E4 and VA RNA in helper viruses is provided. The three plasmids are co-transfected into a HEK-293 cell line, and then can be packaged to form infectious recombinant adeno-associated virus particles. The recombinant adeno-associated virus can stably express foreign genes in cells, and AAV causes a relatively mild immune response in the body compared with other viruses. AAV has found widespread use as a viral vector in gene therapy, prophylaxis and therapeutic vaccines and in basic research.

The aforementioned embodiment of the present invention utilizes eukaryotic expressed ASFV P54 protein as immunogen, selects monoclonal antibody which can effectively neutralize ASFV virus, obtains the sequence of the neutralizing antibody, and clones the neutralizing antibody into AAV vector after codon optimization of the coding gene of the neutralizing antibody, which can be used for treating or preventing African swine fever virus, for example, for preparing African swine fever virus vaccine.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORYMANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and Methodsin Molecular BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.