阅读说明：本技术 抗非洲猪瘟病毒p30蛋白单克隆抗体、制备方法及B细胞表位筛选和鉴定 (anti-African swine fever virus p30 protein monoclonal antibody, preparation method and B cell epitope screening and identification ) 是由 张改平 王爱萍 有小娟 姜文静 陈玉梅 丁培阳 刘红亮 王海丽 周景明 祁艳华 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种抗非洲猪瘟病毒p30蛋白单克隆抗体、制备方法及B细胞表位筛选和鉴定,属于基因工程技术领域,本发明设计并构建能够稳定表达ASFV p30蛋白的H22细胞株和HEK293T细胞株,以稳定表达p30蛋白H22细胞作为免疫原免疫小鼠,利用细胞融合技术,以稳定表达p30蛋白HEK293T细胞作为检测原,通过免疫过氧化物酶单层细胞试验,筛选得到抗ASFV p30蛋白的单克隆细胞株10D7,该细胞株产生的单克隆抗体可特异性识别并结合p30蛋白上的～(171)YGTPLKEEEK～(180)至～(176)KEEEKEVVRL～(185)序列区域,有助于了解抗原-抗体之间的相互作用,对ASFV的检测和诊断具有重要意义。(The invention discloses an anti-African swine fever virus p30 protein monoclonal antibody, a preparation method and B cell epitope screening and identification, belonging to the technical field of genetic engineering, wherein the invention designs and constructs an H22 cell strain and an HEK293T cell strain capable of stably expressing ASFV p30 protein, uses a stably expressing p30 protein H22 cell as an immunogen immune mouse, utilizes a cell fusion technology, uses a stably expressing p30 protein HEK293T cell as a detection source, and screens and obtains an anti-ASFV p30 protein monoclonal cell strain 10D7 through an immunoperoxidase monolayer cell test, and the cell strain produces the anti-ASFV p30 protein monoclonal antibodyThe monoclonal antibody can specifically recognize and bind to p30 protein 171 YGTPLKEEEK 180 To 176 KEEEKEVVRL 185 The sequence region is helpful for understanding the interaction between antigen and antibody, and has important significance for the detection and diagnosis of ASFV.)

1. An epitope of African swine fever virus p30 protein, which is characterized in that the epitope is located at position 171-180 and position 176-185 of p30 protein, and the amino acid sequence is shown as SEQ ID No.1 and SEQ ID No. 2.

2. Use of the epitope according to claim 1 for the preparation of a monoclonal antibody against the p30 protein of African swine fever virus.

3. A monoclonal antibody against the African swine fever virus p30 protein, which specifically binds to an epitope of the African swine fever virus p30 protein of claim 1.

4. The monoclonal antibody against African swine fever virus p30 protein according to claim 3, wherein the CDR-H1 of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID No.3, CDR-H2 is the amino acid sequence shown in SEQ ID No.4, CDR-H3 is the amino acid sequence shown in SEQ ID No. 5; and

the CDR-L1 of the light chain variable region of the monoclonal antibody is an amino acid sequence shown by SEQ ID No.6, the CDR-L2 is an amino acid sequence shown by SEQ ID No.7, and the CDR-L3 is an amino acid sequence shown by SEQ ID No. 8.

5. The monoclonal antibody against p30 of claim 4, wherein the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID No. 9.

6. The monoclonal antibody against p30 of claim 4, wherein the variable region of the light chain of the monoclonal antibody is the amino acid sequence of SEQ ID No. 10.

7. A nucleic acid encoding a monoclonal antibody against the African swine fever virus p30 protein according to any one of claims 3-6, wherein the nucleic acid encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.11 and the nucleic acid encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 12.

8. An expression vector comprising the nucleic acid of claim 7, capable of expressing said nucleic acid in a host cell.

9. A host cell comprising the expression vector of claim 8.

10. Use of a monoclonal antibody against the p30 protein of African swine fever virus according to any one of claims 3-6, a nucleic acid according to claim 7, an expression vector according to claim 8 or a host cell according to claim 9 for the manufacture of a medicament for inhibiting or neutralizing the p30 protein of African swine fever virus.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-African swine fever virus p30 protein monoclonal antibody, a preparation method and B cell epitope screening and identification.

Background

African Swine Fever (ASF) is a contact infectious disease with extremely high lethality, which is typically characterized by high fever and multiple organ bleeding swelling and is caused by African Swine Fever Virus (ASFV), and the disease poses serious threats to animal health, food safety, national economy and environment. ASFV is a large nucleoplasmic virus whose genome consists of double-stranded DNA of 170-190kb, variable regions at both ends, a conserved region of about 125kb in the middle, 35kb at the left end and 15kb at the right end. These tandem repeats bind double-stranded DNA together by covalent bonds. The genome of this virus contains 151-167 Open Reading Frames (ORFs). ASFV is a complex, multi-layered icosahedral virion, with 68 distinct protein components and 21 host cell proteins associated with the virus identified using mass spectrometry techniques.

Among the structural proteins constituting ASFV, p30 is one of important antigen structural proteins and is expressed at an early stage of virus-infected host cells, and it is a protein encoded by CP204L gene and having a relative molecular weight of 30 kDa. p30 is one of the most important antigen structural proteins, and is involved in the entry of ASFV into cells and in hindering transcription and translation in host cells. Expression of p30 protein is typically observed around 2 to 4 hours after viral infection and then persists throughout the infection cycle. Thus, expression of the protein indicates that the virus has infected the host cell and expression of the viral early protein has begun. P30 is highly immunogenic and therefore is often used as a serological candidate for the diagnosis and monitoring of ASF. Therefore, the development of p30 protein specific monoclonal antibody and the further screening of p30 protein epitope have important significance for the deep research of p30 protein biological functions, and the development of a detection method aiming at ASFV is facilitated.

Disclosure of Invention

The invention aims to provide a monoclonal antibody of an African swine fever virus p30 protein, a preparation method and B cell epitope screening and identification so as to solve the problems in the prior art, and the monoclonal antibody can specifically identify the African swine fever virus p30 protein¹⁷¹YGTPLKEEEK¹⁸⁰To¹⁷⁶KEEEKEVVRL¹⁸⁵Sequence region, which is the first reported B cell epitope.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an epitope of African swine fever virus p30 protein, which is located at the 171-180 and 176-185 positions of p30 protein, and the amino acid sequence of the epitope is shown as SEQ ID No.1 and SEQ ID No. 2.

The invention also provides application of the epitope in preparation of a monoclonal antibody of African swine fever virus p30 protein.

The invention also provides a monoclonal antibody of the African swine fever virus p30 protein, which is specifically combined with the epitope of the African swine fever virus p30 protein.

Furthermore, CDR-H1 of the heavy chain variable region of the monoclonal antibody is an amino acid sequence shown in SEQ ID No.3, CDR-H2 is an amino acid sequence shown in SEQ ID No.4, and CDR-H3 is an amino acid sequence shown in SEQ ID No. 5; and

the CDR-L1 of the light chain variable region of the monoclonal antibody is an amino acid sequence shown by SEQ ID No.6, the CDR-L2 is an amino acid sequence shown by SEQ ID No.7, and the CDR-L3 is an amino acid sequence shown by SEQ ID No. 8.

Furthermore, the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID No. 9.

Furthermore, the variable region of the light chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID No. 10.

The immunogen for preparing the monoclonal antibody is prepared by packaging pseudovirus particles by using three plasmids, namely pLVX-IRES-ZsGreen1-p30, psPAX-2 and pMD2.G, through a liposome transfection method, and transducing the obtained pseudovirus particles into H22 cells to obtain an H22 cell strain capable of stably expressing ASFV p30 protein;

the detection antigen for preparing the monoclonal antibody is prepared by packaging pseudovirus particles by utilizing pTRIP-CMV-Puro-p30, psPAX-2 and pMD2.G three plasmids through a liposome transfection method, and transducing the obtained pseudovirus particles into HEK293T cells to obtain HEK293T cell strains capable of stably expressing ASFV p30 proteins;

then, a mouse is immunized by taking an H22 cell strain which stably expresses ASFV p30 protein as an immunogen, and an HEK293T cell strain which stably expresses ASFV p30 protein is taken as a detection antigen by utilizing a cell fusion technology, and the monoclonal antibody is obtained by screening through an Immunoperoxidase monolayer assay (IPMA) and is named as 10D 7.

The invention also provides a nucleic acid for encoding the anti-African swine fever virus p30 protein monoclonal antibody, wherein the nucleic acid for encoding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.11, and the nucleic acid for encoding the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 12.

The present invention also provides an expression vector comprising the nucleic acid described above, which is capable of expressing the nucleic acid in a host cell.

The present invention also provides a host cell comprising the above expression vector.

The invention also provides application of the monoclonal antibody for resisting the African swine fever virus p30 protein, the nucleic acid, the expression vector or the host cell in preparation of drugs for inhibiting or neutralizing the African swine fever virus p30 protein.

The invention discloses the following technical effects:

the monoclonal antibody for resisting African swine fever virus p30 protein is based on design and construction of an H22 cell strain and an HEK293T cell strain capable of stably expressing ASFV p30 protein. An H22 cell strain which stably expresses ASFV p30 protein is used as immunogen to immunize a mouse, a cell fusion technology is utilized, HEK293T cell strain which stably expresses ASFV p30 protein is used as a detection antigen, a monoclonal cell strain 10D7 which resists the ASFV p30 protein is obtained by screening through Immunoperoxidase monolayered cell assay (IPMA), and a monoclonal antibody produced by the cell strain can specifically recognize and combine with the monoclonal cell strain 10D7 of p30 protein¹⁷¹YGTPLKEEEK¹⁸⁰To¹⁷⁶KEEEKEVVRL¹⁸⁵Region, this is the first reported B cell epitope.

The H22 cell strain which stably expresses p30 protein is used as immunogen to immunize a mouse, HEK293T which stably expresses p30 protein is used as a detection source, and the result shows that the titer of the obtained monoclonal antibody can reach 1:6400, so that the monoclonal antibody can be used for immunizing animals by using a living cell carrier and generating antibodies, can be simultaneously used for a plurality of immunological detection means such as ELISA, IPMA and the like, and has good application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is PCR of p30 target gene;

FIG. 2 is the identification of the recombinant vector pLVX-IRES-ZsGreen1-p 30;

FIG. 3 is the identification of the recombinant vector pTRIP-CMV-Puro-p 30;

FIG. 4 shows the screening of H22 cell line stably expressing ASFV p30 protein;

wherein (a) the subcloned H22 cell mass fluoresces; (b) white light plot of H22 cell mass after subcloning;

FIG. 5 is a screen of HEK293T cell line stably expressing ASFV p30 protein;

FIG. 6 shows that Western Blot verifies the expression of p30 protein in H22 cells;

wherein, M is a protein Marker; H22-pLVX is H22 cells without recombinant plasmids; H22-pLVX-p30 is H22 cell containing recombinant plasmid pLVX-IRES-ZsGreen1-p 30;

FIG. 7 shows that Western Blot verifies the expression of p30 protein in HEK293T cells;

wherein, M is a protein Marker; pTRIP-HEK293T is HEK293T cell without recombinant plasmid; pTRIP-p30-HEK293T is HEK293T cell containing recombinant plasmid pTRIP-CMV-Puro-p 30;

FIG. 8 shows that IFA detects HEK293T cell line stably expressing ASFV p 30;

wherein, (a) the primary antibody is ASFV positive pig serum 1: 1000; (b) the primary antibody is ASFV positive pig serum 1: 2000; (c) HEK293T cells without recombinant plasmid (negative control);

FIG. 9 is a multiple antisera titer graph;

wherein, (1) the first antibody is 1:6400 of the serum of a No.1 mouse; (2) the primary antibody is the serum 1:1600 of the No.2 mouse; (3) nonimmunized mouse serum;

FIG. 10 shows the supernatants of hybridoma cell lines tested with IPMA 1D6, 1E9, 2E7, 10D7 and 9C 7;

wherein PC is mouse positive multiple antiserum; NC is the serum of an unimmunized mouse; BC is a blank control;

FIG. 11 shows Western Blot detection of supernatants from 1D6, 1E9, 2E7, 9C7 and 10D7 hybridoma cell lines;

FIG. 12 shows the results of Peptide-ELISA assays for conjugate polypeptides and positive serum reactivity;

FIG. 13 is a Peptide-ELISA assay for the reactivity of p30 mAbs to conjugated polypeptides;

FIG. 14 shows the results of Peptide-ELISA assays for the reactivity of truncated polypeptides and p30 mAbs.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

EXAMPLE 1 selection and preparation of immunogens and Detector sources

The reported position of ASFV p30 is not clear, while p30 protein is generated in the early stage of virus infection, is involved in virus internalization, and blocks the transcription and translation of host cells; researches report that p30 protein expressed by baculovirus is used for immunizing pigs, and the results show that test pigs can stimulate the body to produce neutralizing antibodies after being immunized, and have a certain immune protection effect on African swine fever infection, but some researches report that anti-p 30 antibody does not have neutralizing protection and cannot resist the attack of virulent strains, so that whether p30 has neutralizing protection or not is controversial at present. Therefore, the invention has great significance for further carrying out the work of ASFV p 30.

The H22 cell is derived from Balb/c mouse, so the mouse can only generate antibody of anti-p 30 protein, but not generate antibody against the cell, which lays a foundation for preparing anti-ASFV p30 mAb by directly immunizing Balb/c mouse with the expression cell as antigen.

The ASFV p30 gene is transfected into HEK293T cells, and the HEK293T cells can stably express the p30 protein because the lentivirus vector can integrate a foreign gene into a cell DNA genome. The expression of the foreign gene in the eukaryotic cell has the advantages that the protein space conformation is similar to that of the natural protein, and the integrity of the antigen structure is kept.

Therefore, the invention designs and constructs an H22 cell strain capable of stably expressing ASFV p30 protein as immunogen, and HEK293T capable of stably expressing p30 protein as detection antigen. Specifically, the preparation of the immunogen and the detection source comprises the following steps:

1. primer design

According to the published CP204L gene sequence information of the ASFV China/2018/Anhui XCGQ strain published by NCBI (http:// www.ncbi.nlm.nih.gov), a Primer p30 gene is designed by using Primer 5.0 software, two enzyme cutting sites of Xba I and BamH I are selected (underlined parts), protective bases (italic parts) are added, and the ASFV China2018Anhui XCGQ-p30 gene is synthesized, wherein the Primer sequence is shown in Table 1:

TABLE 1 primer sequence Listing

PCR amplification of the p30 Gene

PCR amplification was performed using the synthetic p30 gene sequence as a template. The PCR amplification system is as follows:

TABLE 2 PCR amplification System

3. Purification and recovery of PCR products

After amplification, the products were identified by electrophoresis using a 1% nucleic acid gel, the results of which are shown in FIG. 1. And finally, selecting a PCR product with a band close to the target band by using a DNA purification and recovery kit for purification and recovery. The concentration of the recovered DNA solution was determined using Nano Drop 2000c and stored at-20 ℃ until use.

4. Double digestion of target gene and vector

(1) The p30 gene/pLVX-IRES-ZsGreen 1 vector/pTRIP-CMV-Puro vector is subjected to double enzyme digestion, and the enzyme digestion reaction system is shown in the following table 3:

TABLE 3 double digestion reaction System

(2) The components are mixed evenly and then placed on a thermostat at 37 ℃ for enzyme digestion overnight.

5. Recovery of the cleavage products

And (3) recovering the enzyme digestion product by using a gel recovery kit, sucking 2 mu L of solution in a centrifuge tube to measure the concentration of the recovered DNA, and storing at-20 ℃ for later use.

6. Construction of recombinant plasmid

(1) The p30 gene is connected with a pLVX-IRES-ZsGreen1/pTRIP-CMV-Puro vector

The following components were added to the EP tube:

TABLE 4 connection System

The solution was mixed well in the centrifuge tube and placed in a 16 ℃ ligator for overnight ligation.

(2) Transformation of

Sucking 5 mul of the ligation product, adding the ligation product into 100 mul of DH5 alpha competent cells, adding 900 mul of nonresistance LB liquid culture medium, and shake-culturing at 37 ℃ and 220r/min for 50 min; after the culture is finished, centrifuging the centrifugal tube solution at the rotating speed of 4000r/min for 4min at room temperature, discarding supernatant, and re-suspending the thallus precipitate by using 100 mu L of liquid LB culture medium; and uniformly coating the heavy suspension on an LB solid culture medium containing ampicillin resistance, horizontally culturing for 20min at room temperature, and then inversely placing in a constant-temperature incubator at 37 ℃ for overnight culture.

(3) PCR screening positive clone of bacterial liquid

Picking smooth single colony, inoculating the colony in LB liquid culture medium containing ampicillin resistance, culturing at 37 deg.C and 220r/min for 6h, centrifuging bacterial liquid at 12000r/min for 2min at room temperature, discarding supernatant, and re-suspending the precipitate with appropriate amount of 1 × PBS; the heavy suspension is centrifuged at the maximum rotation speed (about 12000r/min) for 2min, and the supernatant is taken as a template of PCR of the bacterial liquid.

The following components were added to the EP tube:

TABLE 5 bacterial liquid PCR reaction system

And mixing the reaction components uniformly, and placing the mixture in a PCR instrument for PCR amplification. After PCR of the bacterial liquid, identifying the product by using 1% nucleic acid gel, wherein the result is shown in figure 2, and a bright single target strip is displayed in the sample adding hole 4; as shown in fig. 3, the wells 3 show a destination band of a size similar to the expected size. A monoclonal strain having a band size corresponding to the target gene of ASFV p30 was selected and sequenced by Shanghai Biometrics.

(4) Acquisition of recombinant plasmid

And selecting a positive strain with successful sequencing, and culturing the positive strain in a liquid LB culture medium containing benzyl resistance at 37 ℃ at 220r/min overnight. The Plasmid pLVX-IRES-ZsGreen1-p30/pTRIP-CMV-Puro-p30 was extracted using the Plasmid Miniprep Plus Purification Kit according to the procedures described in the Kit instructions.

7. Construction of H22/HEK293T cell line for stably expressing p30 protein

(1) Recovery of H22/HEK293T cells

Sucking 9mL of RPMI-1640/DMEM complete culture medium into a sterile centrifuge tube; taking out H22/HEK293T cells, immediately transferring the cells into a constant-temperature water bath kettle at 37 ℃, and gently shaking the freezing and storing tube from time to time; transferring the cells in the cryopreserved tube into a prepared sterile centrifuge tube containing 9mL of RPMI-1640/DMEM complete culture medium; centrifuging the centrifuge tube at 1000r/min at room temperature for 9min, and discarding the supernatant; absorbing 6mL RPMI-1640/DMEM complete culture medium to suspend cells, uniformly mixing, transferring to a cell culture bottle, and adding CO₂Culturing in a cell culture box at 37 ℃; after culturing for about 17H, observing the cell growth state under an inverted fluorescence microscope, carrying out cell passage when the cell density reaches about 75% of the cell bottle bottom which is fully paved, sucking a new 6mL RPMI-1640 complete culture medium, and lightly blowing and beating H22 cells by using an electric pipette for carrying out passage culture; while HEK293T requires discarding the culture medium, washing with 4mL sterile 1 × PBS for two times, adding 500 μ L pancreatin cell digestive juice for warm digestion for 2min, adding 5mL DMEM complete culture medium to stop pancreatin digestion, and transferring the cells with electric pipettorGently blowing and beating for subculture.

(2) Packaging of recombinant lentiviruses

24h before transfection, HEK293T cells were plated at 1X 10⁶Inoculating the cell amount to polylysine-treated 6-well plate, adding DMEM complete culture medium to 2mL, and adding CO₂Culturing in a cell culture box at 37 ℃; observing the growth state and density of the cells after 24h, changing to an Opti-MEM complete culture medium when the density reaches about 80% of the bottom of the hole, and starting transfection after two hours; transfection was performed with reference to Lipofectamine 2000 liposome transfection kit instructions; after 10h of transfection, the transfection compound is discarded, the medium is replaced by an Opti-MEM complete medium, the medium is placed in a cell culture box with the temperature of 37 ℃ and the content of 5% CO2 for continuous culture for 36 to 48h, the growth state of the cells is observed by an inverted fluorescence microscope, the supernatant of the medium, namely the lentivirus suspension, is collected when 80% or more of the cells are cracked and shed, the suspension is 12000r/min, and the suspension is centrifuged for 15min at the temperature of 4 ℃ for standby.

(3) Lentiviral transduction of target cells

One day before transduction, resuscitated and subcultured H22/HEK293T cells were plated at 1X 10⁴The amount of each cell was inoculated into a 96-well cell culture plate; observing the cell state the next day, selecting 20 holes of the cells with good state, and conducting transduction after removing the culture medium; the pLVX-IRES-ZsGreen1-p30/pTRIP-CMV-Puro-p30 lentivirus suspensions were added to 96-well cell culture plates containing H22/HEK293T cells, respectively, at 100. mu.L/well.

(4) No recombinant plasmid transduced H22/HEK293T cells

H22/HEK293T cells were transfected and transduced following the same method as (2) and (3).

(5) Screening of Lentivirally transduced H22 cells

After pLVX-IRES-ZsGreen1-p30 lentivirus suspension is transduced for 24h, RPMI-1640 complete culture medium with 100 mu L/hole is supplemented, and the green fluorescent protein expression condition is observed under an inverted fluorescent microscope after the continuous culture is carried out for 24 h; and selecting cell pores with more green fluorescent protein expression and brighter fluorescence intensity for enlarged culture. Filtering the mixed cell suspension with sterile nylon net, loading onto BD FACS Aria III instrument, and screening cells with green fluorescenceThen sorting and collecting green fluorescent protein positive cells in 96-well cell culture plate (containing 200. mu.L/well RPMI-1640 complete medium), 2 cells per well, culturing at 37 ℃; after the obtained green fluorescent protein-positive monoclonal cell masses were blown off, the cells were diluted with RPMI-1640 complete medium according to the counting results by the limiting dilution method, cell suspensions having dilution of 20 cells/mL and 10 cells/mL were added to 1-6 columns and 7-12 columns, respectively, of a 96-well cell culture plate, and added to the 96-well cell culture plate in a volume of 100. mu.L per well, and the subcloned cells were placed at 37 ℃ in 5% CO₂The cells are cultured in a cell incubator and are subcloned twice continuously until a single cell mass with green fluorescent protein positive rate close to 100% is screened out, the obtained positive clone cells are named as pLVX-IRES-ZsGreen1-p30-H22, the result is shown in figure 4, the green fluorescent protein positive rate of the pLVX-IRES-ZsGreen1-p30-H22 cell strain is about 94.9%, and the stability of green fluorescent protein expression is verified.

(6) Screening of Lentivirally transduced HEK293T cells

After the pTRIP-CMV-Puro-p30 lentivirus suspension is transduced for 24h, the lentivirus suspension of a 96-well plate is discarded, puromycin with different amounts is respectively added into a DMEM complete culture medium until the final concentration is 2, 4, 8, 16, 32 and 64 mu g/mL, and CO is added₂Culturing in a cell culture box at 37 deg.C for about 6-8 days; the growth status of cells containing puromycin at various concentrations was observed daily under an inverted fluorescence microscope and screened for approximately 6-8 days. Adding a new complete culture medium containing the puromycin DMEM after liquid change or passage during screening; after 1 week, the negative cells are in a circular or suspension state and the positive cells are in an adherent state, and subcloning is carried out after the adherent cells are subjected to expanded culture and passage; until cell masses were selected which remained adherent to cells and could be stably passaged in DMEM complete medium containing puromycin, the obtained positive clone cells were named pTRIP-CMV-Puro-p30-HEK293T, and as a result, the negative cells were in a round or suspended state and the positive cells were in an adherent state, as shown in FIG. 5.

8. Lysis of target cells

Expanded pTRIP-CMV-Puro-p30-HEK293T/pTRIP-CMV-Puro-HEK293T cells were discardedAfter the culture medium is filled, 2mL of sterile 1 × PBS is used for gently washing twice, 1mL of pancreatin cell digestive juice is added into a cell culture bottle, the cell digestive juice is placed at room temperature for digestion for 2min, 5mL of DMEM is finally added to completely culture the pancreatin to stop digestion, an electric pipettor is used for gently blowing the pancreatin to a single cell, and the cell is transferred into a sterile centrifuge tube after counting; directly blowing the amplified cells of pLVX-IRES-ZsGreen1-p30-H22/pLVX-IRES-ZsGreen1-H22 to single cells by using an electric pipette, transferring the cells to a sterile centrifuge tube after counting the cells, centrifuging for 9min at 1100r/min, discarding supernatant, and collecting cell precipitates; taking 1mL of 1 XPBS to fully resuspend the collected cells, centrifuging the resuspension solution at 12000r/min at 4 ℃ for 10min, collecting supernatant, and removing cell debris; before adding RIPA lysate, phenylmethylsulfonyl fluoride was added in advance at a final concentration of 1mmoL/mL, per 1X 10⁶Adding 100 μ L RIPA lysate into each cell, mixing lysate and cell mixture, standing on ice for lysis for 5-10min, centrifuging at 4 deg.C at 12000r/min for 15min, and collecting supernatant; and (3) taking 40 mu L of collected supernatant, subpackaging the collected supernatant into a centrifuge tube, adding 10 mu L of 5 Xloading Buffer, then putting the centrifuge tube into a water bath pot, boiling for 5min, centrifuging for 1min at 12000r/min, and analyzing the expression effect of the p30 protein through a Western Blot test, wherein the result is shown in figure 6 and figure 7, and a lane H22-pLVX-p30/pTRIP-p30-HEK293T shows a single target band, so that the recombinant p30 protein obtained by the invention has good immunoreactivity.

9. Indirect immunofluorescence assay

The day before the indirect immunofluorescence assay, the selected pTRIP-CMV-Puro-p30-HEK293T cells were plated at 1X 10 cells per well⁴The amount of individual cells was seeded in 96-well cell culture plates; observing the density of a 96-well cell culture plate on the next day of cell spreading, slightly discarding the culture medium in each well by using a pipette when the density of each well reaches about 80%, adding 100 mu L/well paraformaldehyde solution to fix the cells, standing at room temperature for 15min, discarding the fixing solution, washing with 1 XPBS for 3 times, adding 100 mu L/well 0.1% triton X-100, standing at room temperature for 10min, discarding, and finally washing with 1 XPBS for 3 times; diluting African swine fever virus positive pig serum with 5% skimmed milk at a ratio of 1:2000, and adding 100 μ L/well to 96-well cell culture plates, incubated for 1h in a 37 ℃ incubator, and then washed 5 times with PBST; after diluting Fluorescein Isothiocyanate (FITC) labeled goat anti-pig IgG 1:500 with 5% skim milk, 100. mu.L/well was added to a 96-well cell culture plate, incubated at 37 ℃ for 1h, and then washed 5 times with PBST; as shown in FIG. 8, when 100. mu.L of 1 XPBS was added to each well and target cells HEK293T were stably transduced with pTRIP-CMV-Puro-p30, respectively, pTRIP-p30-HEK293T (pTRIP-CMV-Puro-p30-HEK293T) contained a target gene that could recognize and bind to a primary antibody (ASFV-positive pig serum), and thus green fluorescence was observed under an inverted fluorescence microscope, whereas pTRIP-HEK293T (pTRIP-CMV-Puro-HEK293T) contained no target gene that could not express a target protein, and recognized a primary antibody, and thus green fluorescence was not observed.

EXAMPLE 2 preparation of monoclonal antibodies

1. Animal immunization

2 female Balb/c mice of 6-8 weeks old were selected. At 1 × 10⁵The number of each cell was immunized by 500. mu.L, and H22 cells (treated) expressing p30 protein were intraperitoneally immunized 4 times at 3-week intervals. Blood was collected from the tail of the mice 1 week after the third immunization, and serum was isolated and titer was measured, using the non-immunized mice as negative control.

IFA determination of potency of mouse serum

The titer of the multiple antiserum of mice after three-week immunization was measured, and as shown in fig. 9, the titer of the multiple antiserum of mouse No.1 was 1:6400, and the titer of the serum of mouse No.2 was 1: 1600.

3. Preparation of feeder cells

Killing a well-developed Kunming mouse by introducing a neck, and fixing the mouse on an anatomical disc by using a pin; sucking pre-cooled 100mL HAT culture medium and transferring to a sterile plate; sucking about 15mL of HAT selection medium from a sterile plate, and slowly injecting the HAT selection medium into the abdominal cavity of the mouse; then extracting the HAT selective culture medium injected into the abdominal cavity of the mouse by using an injector, transferring the HAT selective culture medium into a prepared sterile plate filled with the HAT selective culture medium, and uniformly mixing; mixing ofThe uniform cell suspension is evenly dripped into a 96-hole cell culture plate and is put at 37 ℃ and 5 percent CO₂Culturing in a cell culture box.

4. Preparation of splenocytes

Collecting blood from the orbit of the hyperimmunized experimental mouse, collecting the blood in an EP tube, and collecting serum as a positive control for screening monoclonal antibodies subsequently for later use; the mice were sacrificed by cervical dislocation with the abdomen facing up and fixed on a dissecting plate; taking 3-6mL of the preheated GNK solution, wetting a 200-mesh nylon net which is sterile and fixed on a 50mL beaker; carefully remove the spleen, place it on a 200 mesh sterile nylon mesh and grind it with scissors, add GNK wash to rinse the grind, allow the spleen single cells to filter through the mesh into a beaker; transferring the spleen cell resuspension in the beaker into a 50mL sterile centrifuge tube, and placing the centrifuge tube into a centrifuge for centrifugation for 10min at 1000 r/min; the upper GNK wash was discarded, 20mL of GNK solution was added to resuspend the cells thoroughly, and the cells were aspirated and counted.

5. Cell fusion

Selecting round SP2/0 cells in logarithmic phase, sucking proper amount of complete culture medium RPMI-1640 to blow semi-adherent SP2/0 cells down for mixing, transferring into a 50mL sterile centrifuge tube, and centrifuging in a centrifuge for 10min at 1000 r/min; discarding the upper layer culture medium, taking 20mL of the preheated GNK re-suspended cell mass, counting cells, mixing splenocytes and SP2/0 cells according to the proportion of 5:1-10:1, placing the mixture into a centrifuge, and centrifuging for 10min at 1000 r/min; discarding the supernatant, putting the cell mixture into a 37 ℃ water bath, dropwise adding 1mL of PEG 1500 fusion agent, and then standing for 90s under the condition of the 37 ℃ water bath; sucking 15mL of GNK lotion to terminate the fusion reaction, then transferring the fusion reaction solution to a water bath environment at 37 ℃ for stabilization for 5min, supplementing GNK lotion until the total volume is 40mL, and centrifuging the solution at 1000r/min for 10 min; discarding the supernatant, adding 120mL of preheated HAT selection medium, resuspending and mixing the cells uniformly; dispersing the fused cell suspension 100. mu.L/well into 96-well cell culture plate paved with feeder cells, and adding CO₂The cell culture box is used for culturing at 37 ℃.

6. Screening of hybridoma cell-positive wells

On day 5 after cell fusion, 60 μ L/well of pre-warmed HAT selection medium was replenished in the fused plates; observing the growth state of the cell mass about 8 days after fusion, and performing half liquid exchange on the hole of the hybridoma cell mass by using HAT selective culture medium; and 2-3 days after the liquid change, when the density of the hybridoma cells in the hole is more than 40% of the density of the hybridoma cells at the bottom of the hole, sucking the culture solution of the hybridoma cell mass as a primary antibody, taking the eyeball serum collected before fusion as a positive control, taking the serum of an unimmunized mouse as a negative control, and screening the positive clone by using an IPMA method.

7. Subcloning of Positive hybridoma cells

Performing subcloning for the first time, performing feeder cell preparation one day in advance, and changing HAT culture medium into HT culture medium; gently blowing and uniformly mixing cells needing subcloning under an aseptic condition, mixing the cells with trypan blue with a proper concentration, and accurately counting the living cells; according to the results of cell counting, the diluted cell suspension was added to a feeder cells-plated plate by a limiting dilution method using HT medium as a diluent, the diluted cell suspension was added to a feeder cells-plated 96-well cell culture plate in a volume of 100. mu.L per well, and the subcloned cells were placed at 37 ℃ in a 5% CO atmosphere₂Enabling the culture medium to grow adherent in an incubator; culturing for about 7-8 days, observing the first subcloned cells under an inverted fluorescence microscope, and selecting the holes with only 1 cell mass for positive screening; and carrying out 24-hole amplification culture on the positive hybridoma cell mass, continuously subcloning for 2-3 times, gradually replacing the HT culture medium with an RPMI-1640 complete culture medium until the obtained monoclonal hybridoma cell strain can stably secrete the required antibody, and detecting cell supernatants of the hybridoma cell strains 1D6, 1E9, 2E7, 10D7 and 9C7 by an IPMA method by using pTRIP-CMV-Puro-p30-HEK293T cells as a detection source after two times of subcloning so as to react with HEK293T cells stably expressing p30 protein, wherein the result is shown in figure 10.

8. Western Blot validation of positive hybridoma cells

pTRIP-CMV-Puro-p30-HEK293T/pTRIP-CMV-Puro-p30-HEK293T was used as a sample of SDS-PAGE, and the primary antibody dilution was replaced with a 1:100 dilution of positive hybridoma cell supernatant, as shown in FIG. 11, lane pTRIP-HEK293T was HEK293T cells without recombinant plasmid, and lane RIP-p30-HEK293T was recombinant plasmid containing the target gene. The Western Blot results showed that the 1D6, 1E9, 2E7, 10D7 and 9C7 monoclonal antibodies reacted strongly with the cell lysis supernatant but not with the pTRIP-CMV-Puro-HEK293T lysis cell supernatant, indicating that these monoclonal antibodies can recognize linear epitopes of the p30 protein.

9. Expanded culture and cryopreservation of hybridoma cells

The positive hybridoma cells obtained by identification are transferred to a 24-hole cell culture plate in an expanding culture mode, and the cells can be frozen and stored when the number of the cells is about 80 percent.

Sucking 5mL of RPMI-1640 complete culture medium, blowing and beating the positive hybridoma cells after the expanded culture, transferring the positive hybridoma cells into a sterile centrifuge tube, centrifuging at the room temperature of 1000r/min for 10min, and discarding the supernatant; adding 1mL of cell frozen stock solution into a centrifuge tube to resuspend cells, fully and uniformly mixing, transferring the cell suspension into a 2mL frozen stock tube, labeling information such as the name and date of the frozen stock cells on the wall of the frozen stock tube, standing for 15-25min at 4 ℃, standing for 2h at-20 ℃, standing for 24h in a refrigerator at-80 ℃, performing gradient cooling, and transferring into liquid nitrogen for long-term storage.

10. Polyclonal antibody variable region genes and sequences

The heavy chain variable region and the light chain variable region of the monoclonal antibody have amino acid sequences shown in SEQ ID No.9 and SEQ ID No.10, and the nucleic acid sequences shown in SEQ ID No.11 and SEQ ID No. 12. Further analyzing to obtain the amino acid sequences of the CDR of the heavy chain variable region of the monoclonal antibody, namely GFSLFNNG, IWRGGAT and AKNGIITTGRTYLARAMDY (SEQ ID No. 3-5); the amino acid sequences of the CDRs in the light chain variable region of the monoclonal antibody are QSVDYDGDSY, AAS, QQSDEDPWT (SEQ ID Nos. 6-8), respectively;

wherein the content of the first and second substances,

SEQ ID No.9：

SGPGLVQPSQSLSITCTVSGFSLFNNGVHWVRQSPGKGLEWLGVIWRGGATGYNAPFMSRLSITKDNSKSQVFFKMNSLHPDDAAIYYCAKNGIITTGRTYARAMDYWGQGTTVTISS；

SEQ ID No.10：

SDMVLMLLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYFCQQSDEDPWTFGWRHQSGNQTGGCCTNCIHLPTIQKAW；

SEQ ID No.11：

tcaggacctggcctagtgcagccctcacagagcctgtccataacctgcacagtctctggtttctcattatttaacaatggtgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatttggagaggtggagccacaggctacaatgcacctttcatgtccagactgagcatcaccaaggacaactccaagagccaagttttctttaaaatgaacagtctgcatcctgatgacgctgccatatactactgtgccaaaaatggaatcattacgacgggacgcacctatgctagggctatggactactggggccaagggaccacggtcacgatctcctca；

SEQ ID No.12：

tcggacatggtcctcatgttgctgctgctatgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcatccaatctagaatctgggatccccgccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctatttctgtcaacaaagtgatgaggatccgtggacgttcggctggaggcaccaatctggaaatcaaacgggcggatgctgcaccaactgtatccatcttcccaccatccagaaagcttgg。

example 3 identification of epitope recognized by monoclonal antibody

1. Sequence homology alignment

The sequence of ASFV p30 protein (Access: AYW34064.1) was obtained from NCBI (http:// www.ncbi.nlm.nih.gov), and the sequences were analyzed by alignment using MegAlign analysis tool.

2. Polypeptide synthesis

After sequence alignment and analysis, the sequence of ASFV p30 protein is obtained from NCBI, and the clone sequence with the advantage of p30 protein or clone sequence with high homology is sent to Gill bioscience for polypeptide synthesis and identification according to Table 6.

TABLE 6 truncated Synthesis protocol for the full-Length amino acid sequence of the p30 protein

3. Conjugation of polypeptides

Coupling the synthesized polypeptide with Bovine Serum Albumin (BSA) by glutaraldehyde method, and packaging the obtained synthetic product in an ultra-low temperature refrigerator at-80 deg.C.

Peptide-ELISA assay

The reactivity of the conjugated polypeptide with ASFV positive pig serum, ASFV p30 positive mouse serum and the screened ASFV p30 monoclonal antibody is determined by a Peptide-ELISA method by using the conjugated polypeptide as a coating antigen.

The results are shown in figure 12, and the polypeptides P2, P5, P6, P10 and P12 can react with the serum of an ASFV P30 positive mouse; and the polypeptides P2, P5, P6, P10, P12 and P13 react with ASFV-positive pig serum. The polypeptides P2, P5, P6, P10, P12 and P13 are immunodominant dominant region peptide fragments of the P30 protein.

The Peptide coupled to BSA was coated on an ELISA plate, and Peptide-ELISA was performed using the supernatant of the selected monoclonal antibody against P30 and the Peptide, and the results are shown in FIG. 13, in which P2, P5 and P6 were recognized by 9C7 mAb, P6 by 2E7 mAb and P12 by 10D7 mAb.

Further positioning of B-cell epitope amino acid sequence of ASFV p30 protein

(1) Peptide fragment truncation synthesis scheme

According to the result of the primary epitope mapping, the identified positive synthetic peptide is further truncated and synthesized, P6 is further truncated into P6-1, P6-2 and P6-3, P12 is further truncated into P12-1, P12-2 and P12-3, and the truncation scheme is shown in Table 7.

TABLE 7 amino acid residue sequences of truncated polypeptides

(2) Conjugation of truncated Polypeptides to BSA

The synthetic truncated polypeptides were conjugated to BSA.

(3) ELISA detection of truncated polypeptide and monoclonal antibody

The successfully conjugated polypeptides were identified by Peptide-ELISA. As shown in FIG. 14, the results showed that P was recognized by 10D7 mAb12-2 and P12-3, and its epitope is positioned on¹⁷¹YGTPLKEEEK¹⁸⁰(SEQ ID No.1) to¹⁷⁶KEEEKEVVRL¹⁸⁵(SEQ ID No.2) region, which is the first reported B cell epitope.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Zhengzhou university

HENAN ZHONGZE BIOLOGICAL ENGINEERING Co.,Ltd.

<120> African swine fever virus p30 protein monoclonal antibody, preparation method and B cell epitope screening and identification

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Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Arg Gly

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Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu His

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Pro Asp Asp Ala Ala Ile Tyr Tyr Cys Ala Lys Asn Gly Ile Ile Thr

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tcaggacctg gcctagtgca gccctcacag agcctgtcca taacctgcac agtctctggt 60

ttctcattat ttaacaatgg tgtacactgg gttcgccagt ctccaggaaa gggtctggag 120

tggctgggag tgatttggag aggtggagcc acaggctaca atgcaccttt catgtccaga 180

ctgagcatca ccaaggacaa ctccaagagc caagttttct ttaaaatgaa cagtctgcat 240

cctgatgacg ctgccatata ctactgtgcc aaaaatggaa tcattacgac gggacgcacc 300

tatgctaggg ctatggacta ctggggccaa gggaccacgg tcacgatctc ctca 354

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tcggacatgg tcctcatgtt gctgctgcta tgggttccag gctccactgg tgacattgtg 60

ctgacccaat ctccagcttc tttggctgtg tctctagggc agagggccac catctcctgc 120

aaggccagcc aaagtgttga ttatgatggt gatagttata tgaactggta ccaacagaaa 180

ccaggacagc cacccaaact cctcatctat gctgcatcca atctagaatc tgggatcccc 240

gccaggttta gtggcagtgg gtctgggaca gacttcaccc tcaacatcca tcctgtggag 300

gaggaggatg ctgcaaccta tttctgtcaa caaagtgatg aggatccgtg gacgttcggc 360

tggaggcacc aatctggaaa tcaaacgggc ggatgctgca ccaactgtat ccatcttccc 420

accatccaga aagcttgg 438