CN117843732A

CN117843732A - African swine fever virus p30 protein related linear B cell epitope and application thereof

Info

Publication number: CN117843732A
Application number: CN202410048808.0A
Authority: CN
Inventors: 张改平; 吴亚楠; 田盼盼; 王梦翔; 孙卓雅; 宋金星; 孙俊如; 周蕾; 张二芹; 庄国庆; 杜永坤
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-04-09

Abstract

The application belongs to the technical field of animal vaccine preparation, and particularly relates to a linear B cell epitope related to an African swine fever virus p30 protein and application thereof. The linear B cell epitope related to the African swine fever virus p30 protein is a polypeptide with 7 amino acid residues, and the specific amino acid sequence is shown in SEQ ID No.1, and specifically comprises: HNFIQTI. Analysis of the identified B cell epitope information shows that the epitope is conserved across all reference ASFV lines in different regions of china, including the widely distributed highly pathogenic strain Georgia 2007/1 (NC 044959.2). Generally, based on the experimental results, a certain good technical foundation can be laid for the preparation of related African swine fever detection reagents and the development of related vaccines.

Description

African swine fever virus p30 protein related linear B cell epitope and application thereof

Technical Field

The application belongs to the technical field of animal vaccine preparation, and particularly relates to a linear B cell epitope related to an African swine fever virus p30 protein and application thereof.

Background

African Swine Fever (ASF) is an acute infectious disease caused by African Swine Fever Virus (ASFV). ASFV is a large double-stranded DNA virus with complex structure, the genome length of different strains is between 170-193kb, there are 151-190 open reading frames, encoding 150-200 proteins, including at least 54 structural proteins. Because of its high infectivity and high lethality, it has a serious impact on pig industry and meat market supply in various countries. ASFV has complex structure and immune evasion mechanism, huge genome and genetic diversity, and has difficulty in controlling African swine fever, and effective vaccine or antiviral strategies are still lacking at present. Therefore, there is an urgent need to develop an immunological function study of the important antigen proteins of ASFV, and analyze and identify viral protein antigens with immunoprotection and serological diagnostic value.

The p30 protein is taken as an important structural protein expressed in the early replication of ASFV, and participates in the adsorption and internalization process of viruses; the p30 protein is also one of the most antigenic proteins in ASFV and can elicit the production of neutralizing antibodies in infected animals. Therefore, in the prior art, p30 protein is used as a target for detecting and diagnosing African swine fever, and p30 (or combined with other structural proteins) is used for developing and using related African swine fever-resistant vaccines.

Because of the significance of the p30 protein in the African swine fever virus structure, the p30 protein is subjected to intensive research and has important technical significance for analyzing the action mechanism of viruses and promoting vaccine development.

Disclosure of Invention

Based on monoclonal antibody technology application, the application aims at providing a linear B cell epitope related to African swine fever virus p30 protein, thereby laying a certain technical foundation for African swine fever virus detection and related vaccine development.

The technical scheme adopted by the application is described in detail below.

The linear B cell epitope related to the African swine fever virus p30 protein is a polypeptide with 7 amino acid residues, and the specific amino acid sequence is shown in SEQ ID No.1, and specifically comprises: HNFIQTI, namely: his-Asn-Phe-Ile-Gln-Thr-Ile.

The linear B cell epitope related to the African swine fever virus p30 protein is applied to preparation of African swine fever detection reagents.

The linear B cell epitope related to the African swine fever virus p30 protein is applied to preparation of African swine fever vaccines.

The gene sequence for encoding the African swine fever virus p30 protein is shown as SEQ ID No.2, and is specifically as follows (585 bp): ATGGACTTCATCCTGAACATCTCTATGAAAATGGAAGTTATCTTCAAAACCGACCTGCGTTCTTCTAGTCAGGTTGTCTTCCACGCAGGTTCTCTGTACAACTGGTTCTCTGTTGAAATCATCAACTCTGGTCGTATCGTTACCACCGCTATCAAAACCCTGCTGTCTACCGTTAAATACGACATCGTTAAATCTGCTCGTATCTACGCTGGTCAGGGTTACACCGAACACCAGGCTCAGGAAGAATGGAACATGATCCTGCACGTTCTGTTCGAAGAAGAAACCGAATCTTCTGCTTCTTCTGAAAACATCCACGAAAAAAACGACAACGAAACCAACGAATGCACCTCTTCTTTCGAAACCCTGTTCGAACAGGAACCGTCTTCTGAAGTTCCGAAAGACTCTAAACTGTACATGCTGGCTCAGAAAACCGTTCAGCACATCGAACAGTACGGTAAAGCTCCGGACTTCAACAAAGTTATCCGTGCTCACAACTTCATCCAGACCATCTACGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTTGTTCGTCTGATGGTTATCAAACTGCTGAAAAAAAAATAA.

A monoclonal antibody for resisting African swine fever virus p30 protein comprises a heavy chain and a light chain, wherein the heavy chain is of an IgG1 subclass, and the light chain is of a kappa type;

the heavy chain amino acid sequence (116 aa) is specifically as follows:

EVQFIESGGGLVQPKGSLKLSCAASGFTFNTFAMNWVRQAPGKGLEWIARIRSKSNNYATY YADSVKDRFTISRDDSQSMVYLQMNNLKTEDTAIYYCVRHGYDYWGQGTTLTVSS；

the amino acid sequences of the CDRs of the heavy chain variable region are GFTFNTFA, IRSKSNNYAT, VRHGYDY (corresponding nucleotide sequences: GGATTCACCTTCAATACCTTCGCC, ATAAGAAGTAAAAGTAACAATTATGCAACA, GTGAGACATGGTTATGACTAC, respectively);

the light chain amino acid sequence (106 aa) is specifically as follows:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSHKRWIYDTSKLASGVPGRF SGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRTFGGGTKLEIK；

the amino acid sequences of the CDRs of the light chain variable region are SSVSY, DTS, QQWSSNPRT (corresponding nucleotide sequences: TCAAGTGTAAGTTAC, GACACATCC, CAGCAGTGGAGTAGTAACCCACGGACG, respectively);

the coding nucleotide sequence (348 bp) corresponding to the heavy chain is specifically as follows:

GAGGTGCAGTTTATTGAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCGTTGAAACT

CTCATGTGCAGCCTCTGGATTCACCTTCAATACCTTCGCCATGAACTGGGTCCGCCAGGC

TCCAGGAAAGGGTTTGGAATGGATTGCTCGCATAAGAAGTAAAAGTAACAATTATGCAA

CATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCA

TGGTCTATCTGCAAATGAACAACTTGAAAACTGAAGACACAGCCATATATTACTGTGTGAGACATGGTTATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

the coding nucleotide sequence (318 bp) corresponding to the light chain:

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCAC

CATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCTG

GCACCTCCCACAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGGT

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGC

TGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

the monoclonal antibody of the African swine fever virus p30 protein can be particularly used for an African swine fever detection reagent or used for preparing and applying African swine fever vaccine related products.

Because the development of African swine fever vaccine still has higher technical difficulty, the early diagnosis of ASFV still has very important technical significance for the prevention and control of African swine fever. In the application, considering that ASFV p30 protein is one of the antigens with the most immunodominance, firstly, the coding sequence is optimized based on the aim of improving the expression level when preparing p30 protein by recombination, and the recombinant ASFV p30 protein is prepared. Using this recombinant ASFV p30 protein, the inventors immunized BALB/c mice and screened hybridoma cell lines secreting specificity McAb (Monoclonal Antibody) (1B 4G2-4, subtype of antibody: igG 1/kappa-type). The related experimental results show that the secreted McAb can specifically bind to ASFV Pig/HLJ/2018 strain. Further, the identification result of the related binding epitope information shows that the binding sites of the linear B cell epitope are as follows: ¹⁶⁴ HNFIQTI ¹⁷⁰ 。

further analysis of the identified B cell epitope information shows that the epitope is conserved in all reference ASFV lines in different regions of china, including the widely distributed highly pathogenic strain Georgia 2007/1 (NC 044959.2). Generally, based on the experimental results, a certain good technical foundation can be laid for the preparation of related African swine fever detection reagents and the development of related vaccines.

Drawings

FIG. 1 is an illustration of the identification of purified products of recombinant p30 proteins of ASFV and the immunogenicity of the recombinant p30 proteins; wherein:

a is the SDS-PAGE identification result of the recombinant p30 protein prepared by expression; wherein M: protein markers (10 kDa to 180 kDa); lane 1: purified p30 protein;

b is the result of immunoblotting analysis on the purified p30 protein, wherein lane M: protein markers (10 kDa to 180 kDa); lane 1: non-induced E.coli lysate; lane 2: purified p30 protein;

FIG. 2 shows experimental results related to the preparation process of the prepared monoclonal antibody; wherein:

a is the serum efficacy of p30 protein immunized mice before the preparation of fusion hybridoma cells by indirect ELISA detection;

b is titer determination of McAblB 4G2-4 by ELISA method;

subtype C is McAb lB4G 2-4;

FIG. 3IFA and immunoblotting method for specificity analysis of monoclonal antibody 1B4G 2-4; wherein:

a is IFA detection result for p30 protein; determining the reactivity of McAb1B4G2-4 with ASFV p30 recombinant plasmid in PK15 cells using IFA assay; DAPI-stained nuclei blue p30 transfected PK15 cells were incubated with McAb1B4G2-4, followed by FITC-conjugated goat anti-mouse IgG (1:500)

B is McAb lB4G2-4 specifically combined with p30 protein, and the figure shows that: lane 1: negative control lane 2 with the unrelated protein pB 602L: p30 protein;

c is McAb lB4G2-4 strain capable of specifically binding ASFV HLJ/2018; lane M: protein labeling; lane 1: PAM cells not infected with ASFV pig/HLJ/2018 strain served as blank control; lane 2, pig alveolar macrophage inactivation sample of ASFV swine/HLJ/18 strain;

FIG. 4 shows the results of a titer assay for McAblB 4G2-4 by blocking/competitive ELISA; wherein:

a negative anti-ASFV serum for determining baseline OD of ELISA ₄₅₀ A value;

b is an absorbance value statistical result, and data in the graph are expressed by mean ± standard deviation; the data is determined by t-test, and P is less than 0.01;

FIG. 5 is a schematic representation of the design of truncated overlapping short peptides and cloning of a series of truncated fragments of p30 into pGEX-6p-1 and expression as GST tagged fusion proteins, preliminary epitope identification by Western blotting. In the figure:

a is a schematic representation of a truncated overlapping short peptide design across the p30 protein;

b is a Western immunoblotting detection result performed by using McAb1B4G2-4 after the first time of truncation;

c is the Western blotting detection result by using McAb1B4G2-4 after the second time of truncation;

FIG. 6 is a precise localization of linear epitopes recognized by McAb1B4G 2-4; in the figure:

a is a related polypeptide sequence artificially synthesized after further shortening;

b is a dot blot analysis experimental result aiming at different peptide fragments, and PBS is used as negative control;

c is the experimental result of dot blot analysis of the further synthesized polypeptide P15;

FIG. 7 is a sequence conservation analysis and spatial structure analysis of the identified linear B cell epitopes; in the figure: a is a conservation analysis result based on multi-sequence alignment, and a square frame is marked as ¹⁶⁴ HNFIQTI ¹⁷⁰ A sequence;

b, using PyMOL to draw the skeleton of ASFV p30 protein; the structure of the p30 protein in the figure is predicted by the Phyre2 on-line server, the (under color view) surface is a cyan backbone, ¹⁶⁴ HNFIQTI ¹⁷⁰ the sequence (blue in color view) is shown on the p30 protein.

Detailed Description

The present application is further illustrated below with reference to examples. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.

Biological material:

ASFV positive standard serum from chinese veterinary collection of bacterial cultures (CVCC, beijing, china);

ASFV negative serum, hybridoma cells (SP 2/0), BALB/c mice, etc. are common and commonly used biological materials in the art, and can be obtained from public sources, and the applicant (inventor) belongs to a professional research institution, so that related biological materials are purchased and stored for a long time;

the pCMV-3xFlag plasmid and PK-15 cells (pig kidney cells) are common and commonly used experimental materials in the prior art, and can be obtained from public channels, and the work unit of the inventor is taken as a professional teaching and research institution, and related experimental materials are purchased and stored for a long time;

QuickAntibody-Mouse5W, available from Souzhou Bochu immunotechnology Co., ltd. (Suzhou, china);

inactivated ASFV Pig/HLJ/2018 strain infects Porcine Alveolar Macrophages (PAMs), provided by Harbin veterinary research; experimental reagent:

FITC-labeled goat anti-mouse IgG, product of Sanying Biotechnology Co., ltd (Wuhan).

Example 1

The main technical ideas of the application are as follows: because the p30 protein has better antigen immunity, the p30 protein is utilized to immunize a mouse to screen and obtain a hybridoma cell line capable of generating monoclonal antibodies, and related antigen epitopes are further identified, so that a foundation is laid for further preparation of African swine fever detection reagents and vaccine preparation.

Considering the technical problem that the effect is uncertain (for example, the expression amount may be low) when the p30 protein is obtained by using the genetic engineering technology to combine with the original p30 protein coding sequence for expression, the inventor further optimizes the gene coding sequence based on the gene sequence of CP204L of the existing ASFV China/2018/Anhui XCGQ isolate strain in the prior art and combines with the characteristics of the subsequent genetic engineering expression strain (namely, the preference of combining with enterobacter codons), and the optimized gene sequence is shown as SEQ ID No.2, and is as follows:

ATGGACTTCATCCTGAACATCTCTATGAAAATGGAAGTTATCTTCAAAACCGACCTGCGTTCTTCTAGTCAGGTTGTCTTCCACGCAGGTTCTCTGTACAACTGGTTCTCTGTTGAAATCATCAACTCTGGTCGTATCGTTACCACCGCTATCAAAACCCTGCTGTCTACCGTTAAATACGACATCGTTAAATCTGCTCGTATCTACGCTGGTCAGGGTTACACCGAACACCAGGCTCAGGAAGAATGGAACATGATCCTGCACGTTCTGTTCGAAGAAGAAACCGAATCTTCTGCTTCTTCTGAAAACATCCACGAAAAAAACGACAACGAAACCAACGAATGCACCTCTTCTTTCGAAACCCTGTTCGAACAGGAACCGTCTTCTGAAGTTCCGAAAG ACTCTAAACTGTACATGCTGGCTCAGAAAACCGTTCAGCACATCGAACAGTACGGTAAAGCTCCGGACTTCAACAAAGTTATCCGTGCTCACAACTTCATCCAGACCATCTACGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTTGTTCGTCTGATGGTTATCAAACTGCTGAAAAAAAAATAA。

correspondingly, the amino acid sequence of the p30 protein is as follows:

MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK。

based on the optimized gene coding sequence, the inventor entrusts the engineering biological technology limited company (Zhengzhou, china) to synthesize the gene sequence and further clone the gene sequence into a pET-30a plasmid (the recombinant plasmid is named pET-30a-p 30).

Using the plasmid pET-30a-p30, the inventors performed expression preparation and purification of recombinant proteins, and the detailed experimental procedure was outlined below.

(one) transformation and Induction of protein expression

After plasmid pET-30a-p30 is transformed into competent cells of escherichia coli BL21, the correct transformed strain is obtained by screening and identification, and the transformed correct strain is further amplified to OD ₆₀₀ When about 0.6 is included, IPTG (isopropyl-beta-D-1-thiogalactoside) is added to the mixture to obtain the final concentration of 0.2mM, and the mixture is further cultured for 14 hours under the condition of 16 ℃ and 220rpm so as to obtain the His-marked ASFV p30 protein by induction expression.

(II) protein purification

Taking bacterial liquid after the induction culture in the step (one), centrifugally collecting bacterial, washing bacterial precipitate by PBS, and then re-suspending bacterial sludge according to the proportion of 1 gram of bacterial sludge and 10 milliliters of Buffer A (20 mM/L Tris,150mmol/L NaCl,5% glycerol), and carrying out ultrasonic disruption for 80 minutes under ice bath conditions to crack the bacterial; subsequently, the lysate after cell disruption was centrifuged at 12000r/min at 4℃for 1 hour, and the supernatant was collected and purified using Ni-Sepharose 6Fast Flow resin (GE Healthcare). Specific purification operations are referenced as:

the supernatant was applied to a Ni-NTA affinity column, and after the whole sample was applied, buffer A (20 mM/L Tris,150mmol/L NaCl,5% glycerol, 50mM imidazole), buffer B (20 mM/L Tris,150mmol/L NaCl,5% glycerol, 200mM imidazole), and buffer C (20 mM/L Tris,150mmol/L NaCl,5% glycerol, 500mM imidazole) were eluted, wherein buffer A was eluted with 20 column volumes, buffer B was eluted with 10 column volumes, and buffer C was eluted with 10 column volumes.

Finally, the purity and reactivity of the purified proteins were analyzed by SDS-PAGE and Western Blot (WB). The 200mM imidazole eluted solution was sampled using 5X protein loading (ABclonal, chinese Wuhan) (i.e., 5X protein loading was added to p30 protein solution, boiling at 98℃for 10 minutes), and identified by SDS-PAGE, as shown in FIG. 1A, p30 protein was successfully expressed and purified in soluble fraction, and purity was very high at 95% and molecular weight was about 35kDa, which is consistent with the expected results;

meanwhile, the non-induced escherichia coli is used as a negative control, SDS-PAGE gel is transferred to a PVDF membrane, then the PVDF membrane and ASFV standardized positive serum are used as primary antibodies, the primary antibodies are incubated for 1h, a mouse-anti-pig coupling HRP antibody (diluted 1:5000) (Solebao, beijing, china) is used as secondary antibodies, and the secondary antibodies are incubated for 1h at room temperature to develop color analysis results. The related detection results are shown in fig. 1B, and the position of the occurrence of the band on the PVDF membrane incubated with positive serum is about 35KD, which indicates that the prepared ASFV p30 recombinant protein has better reactivity.

(III) measuring the amount of the test substance to be used

In order to facilitate the subsequent detection application, the inventor adopts a chessboard titration method to coat the prepared p30 recombinant protein on a 96-hole ELISA plate, adds diluted serum to be detected (ASFV positive serum sample), and simultaneously takes a mouse-pig-coupled HRP antibody (1:5000 dilution) as a secondary antibody for incubation for 1h; ASFV negative serum was also used as negative control. The optimal antigen coating amount of the prepared p30 protein (antigen) is detected and measured. The correlation results are shown in table 1 below.

TABLE 1 optimal antigen coating concentration and serum dilution experimental results

P: OD value of positive samples; n: OD value of negative samples.

From the above table results, it can be seen that: the difference in OD values between positive and negative sera was greatest (P/N value 11.636) when the dilutions of antigen and serum were 2. Mu.g/mL and 1:200, respectively. Thus, at the subsequent detection application, the applied concentration of antigen was determined to be 200 ng/well (96 well plate) and the serum dilution was 1:200.

Example 2

Based on the purified p30 protein obtained in example 1, BALB/c female mice were immunized further, and monoclonal antibodies were obtained for further analysis by trial preparation, as follows.

Animal immunization

8 week old female BALB/c mice (25. Mu.g each) were vaccinated (i.p.) with the purified p30 protein obtained in example 1, prepared p30 protein was mixed with the Quick-anti-Mouse-5W adjuvant product of Boolong company at a 1:1 mass ratio, and the mice were vaccinated by intramuscular injection on day 0 and day 21, respectively, during which period the mice had free diet;

on day 35, mice with high serum antibody titers were selected by intravenous blood sampling from the tail of mice after immunization, and hyperimmunized (without adjuvant) by intraperitoneal injection of 50 μg of p30 protein for 1 time, with reference to the amounts detected in example 1.

After the end of the boost immunization for 3 days, an indirect ELISA test method is adopted to test the serum titer of the mice, and the result shows that the maximum ratio can be 1:562000 (figure 2A), and the result can lay a good technical foundation for the next cell fusion.

(II) preparing and obtaining monoclonal cell strain and identifying monoclonal antibody

Referring to the conventional operation in the prior art, spleen cells of the mice after the immunization are isolated and obtained in the step (one), and are subjected to cell fusion with SP2/0 cells according to the quantity ratio of 10:l;

and screening positive hybridoma cells capable of secreting the anti-p 30 antibody by adopting an indirect enzyme-linked immunosorbent assay on the fused cells, and carrying out limited dilution subcloning on the screened positive hybridoma cells for 3 times to obtain monoclonal cells secreting the p30 antibody.

The monoclonal cells obtained by the screening were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and further injected into BALB/c mice (female, 8 weeks old), and ascites after injection was collected for 10 to 14 d. Antibody titer in ascites was determined by ELISA. The monoclonal antibodies produced by the p30 protein were then subjected to subtype analysis by reference to the mouse monoclonal antibody subtype identification kit instructions (protection, china). The collected ascites was further purified by Protein A immunoabsorption column chromatography.

The specificity of the monoclonal antibodies (i.e., whether or not they are capable of specifically binding ASFVp30 protein and strain) was further determined by western immunoblotting (reference example one of specific experimental procedures) and indirect IFA, respectively, on the purified monoclonal antibodies.

The final screened hybridoma cell line against ASFV p30 protein was designated lB4G2-4 and the relevant results are shown in fig. 2. Specifically:

based on ELISA detection, the p30 antibody levels in ascites showed high titers, with p 30-primed endpoint titers ranging from 1:5000 to 1:2560000 (fig. 2B).

Subtype analysis showed (FIG. 2C) that the heavy chain subclass of McAblB 4G2-4 was IgG1 and the light chain was kappa type. Further correlation sequencing analysis results show that the amino acid sequences of the heavy chain and the light chain of the monoclonal antibody of the invention are specifically as follows.

Heavy chain amino acid sequence (116 aa):

EVQFIESGGGLVQPKGSLKLSCAASGFTFNTFAMNWVRQAPGKGLEWIARIRSKSNNYATYYADSVKDRFTISRDDSQSMVYLQMNNLKTEDTAIYYCVRHGYDYWGQGTTLTVSS; the amino acid sequences of the CDRs of the heavy chain variable region are GFTFNTFA, IRSKSNNYAT, VRHGYDY, respectively (their corresponding nucleotide sequences: GGATTCACCTTCAATACCTTCGCC, ATAAGAAGTAAAAGTAACAATTATGCAACA, GTGAGACATGGTTATGACTAC, respectively;

light chain amino acid sequence (106 aa):

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSHKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRTFGGGTKLEIK；

heavy chain nucleotide sequence (348 bp):

GAGGTGCAGTTTATTGAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCGTTGAAACT

CTCATGTGCAGCCTCTGGATTCACCTTCAATACCTTCGCCATGAACTGGGTCCGCCAGGC

TCCAGGAAAGGGTTTGGAATGGATTGCTCGCATAAGAAGTAAAAGTAACAATTATGCAA

CATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCA

light chain nucleotide sequence (318 bp):

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCAC

CATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCTG

GCACCTCCCACAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGGT

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGC

according to WB detection results (figure 3), mcAb1B4G2-4 and P30 protein and PAM can generate affinity reaction after ASFV HLJ/2018ASFV strain infection; the IFA result shows that the monoclonal antibody has strict specificity with the transient expression product of ASFV p30 protein in cells, green fluorescence is visible under a microscope, and the control hole transfected with the empty vector is non-fluorescent.

It should be noted that, in the case of immunofluorescence assay (IFA) detection (detection of p30 protein monoclonal antibody specificity), specific procedures are referred to as follows:

firstly, referring to the conventional operation, preparing a pCMV-3xFlag-p30 recombinant plasmid containing a p30 gene (the gene sequence is shown as SEQ ID No. 5);

then, respectively transfecting PK15 cells with a pCMV-3xFlag-p30 recombinant plasmid and a pCMV-3xFlag empty vector plasmid (negative control), and culturing and growing the transfected cells for 24 hours;

after 24h incubation, the medium was discarded and the cells were fixed in 5% paraformaldehyde solution for 30min;

after the fixation is finished, the mixture is washed clean by PBS, is permeabilized by Triton X100 for 15min, and is sealed for 1h;

using p30 monoclonal antibody (1:1 000) as primary antibody, incubating l h at room temperature; meanwhile, taking the positive serum of the mouse as a primary antibody positive control;

subsequently, FITC-labeled goat anti-mouse IgG was used as the secondary antibody;

finally, 4', 6-diamidino-2-phenylindole (DAPI; beyotidme) was stained in the dark for 10 minutes, and then stained with PBS and ddH ₂ O was rinsed three times separately, added with sealer and dried in the dark, and finally inspected using LSM 800 laser scanning confocal microscope (zeiss, germany).

(III) reactivity detection of the prepared monoclonal antibody

To evaluate the generation of antibodies against the p30 epitope in positive anti-ASFV serum, the inventors further performed a blocking/competing enzyme-linked immunosorbent assay based on the ELISA detection principle (i.e., the blocking effect of McAb on positive anti-ASFV serum was determined by blocking enzyme-linked immunosorbent assay with monoclonal antibodies as blocking antibodies). The specific experimental operation and process are referred as follows:

the purified recombinant p30 protein prepared in example 1 was diluted to 200ng/mL using phosphate buffered saline (ph=9.6), added to 96-well plates in an amount of 100 μl per well, and incubated for 1 hour at 37 ℃; after the incubation is finished, the plates are washed three times by TBST and shaken to dryness;

subsequently, the plates were closed with 5% SM plates for 1 hour at room temperature, and then washed three times with TBST;

ASFV positive serum was pooled using 1% BSA in PBS according to 1:1, and then adding 50 mu L of each of the diluted culture supernatant and hybridoma cell culture supernatant into a 96-well plate to serve as a test well; in addition, ASFV negative sera were prepared using 1% BSA in PBS according to 1:1, and then adding 50 mu L of each of the diluted solution and hybridoma cell culture supernatant into a 96-well plate to serve as a negative well; incubation at 37 ℃ for 1 hour;

after incubation was completed, plates were washed three times with TBST and incubated with 5% SM diluted 1:5000hrp conjugated goat anti-mouse IgG for 1 hour at room temperature; after incubation, washing the plates by TBST for three times;

3,3', 5' -tetramethylbenzidine (TMB, solarbio) was added, incubated at room temperature for 10 minutes, and after the incubation was completed, 3mol/L HCl (50. Mu.L/well) was added to stop the reaction;

finally, the optical density value at the wavelength of 450 nm (Tecan 10M multimode microplate reader); each reaction was repeated three times and absorbance values were converted to Percent Inhibition (PI) as follows:

PI (%) = [1- (OD of test sample) ₄₅₀ OD of negative control ₄₅₀ )]×100％。

The correlation results indicate (FIG. 4) that the inhibition rate of McAb to positive anti-ASFV serum is greater than 50%. This result indicates that 1B4G2-4 can block binding of positive anti-ASFV serum to p30 protein. That is, this indicates that the epitope corresponding to 1B4G2-4 is capable of generating potent B cell immunity in ASFV-infected pigs.

Example 3

Based on the results of example 2, the inventors further analyzed and identified the minimal linear B epitope that can be recognized by the monoclonal antibody obtained by the preparation, and the specific cases are described below (the operations are not described in detail, and only the prior art is referred to, and are not described in detail).

To initially map the epitope of 1B4G2-4, first, using a truncation method, the inventors designed three partially overlapping short peptides (P1 to P3), each of 76 amino acids in length (to span the entire length of the P30 protein, as shown in fig. 5);

then, based on the designed truncated fragment, designing a related primer and cloning and recombining a related sequence into a pGEX-6p-1 vector, and then transforming a recombined plasmid into competent cells of escherichia coli BL21 (DE 3) (transforming by a heat shock method); then, positive clone bacteria with correct transformation are selected and cultured until OD ₆₀₀ When about 0.6, IPTG was added at a final concentration of 1M for induction expression (induction at 37 ℃ for 4 hours at 220 rpm); after the induction expression is finished, bacterial liquid is taken for SDS-PAGE electrophoresis detection and identification, so that the correct expression is ensured (the short peptides are expressed as MBP fusion proteins in escherichia coli, and specific operation is only needed by referring to the prior art and is not repeated).

The truncated proteins with correct expression after the above identification were identified using the WB technique using peroxidase-conjugated anti-GST antibodies (Proteintech, china, 1:5000) and monoclonal antibodies prepared in example 2 above.

The results show that 1B4G2-4 can specifically bind to the P3 (119-194 aa) region;

further, the second truncation and recombinant protein expression preparation were performed based on the above results, and Western blot analysis was further performed on the prepared MBP peptide fusion protein, and the final results showed that: 1B4G2-4 recognition region is ¹⁵⁷ FNKVIRAHNFIQTIYGTPLK ¹⁷⁷ 。

It should be noted that, the sequence information of the related primers designed during the preparation of the related recombinant proteins is shown in the following table 2.

TABLE 2 primers designed during protein truncation

Based on the above results, the inventors further developed peptides in order to further accurately map the core sequence of McAb1B4G2-4 linear epitope ¹⁵⁷ FNKVIRAHNFIQTIYGTPLK ¹⁷⁷ Eight overlapping short peptides (P7-P14, shown in FIG. 6) are cut from the N end, related polypeptide sequences are further synthesized artificially, and meanwhile, experimental detection is carried out on the minimum B cell epitope condition of the synthesized peptides by adopting a dot blot method, and the result is shown in FIG. 6.

Analysis can be seen: P8-P14 were recognized by McAb1B4G2-4, but further synthesized P15 was not reactive with McAb1B4G 2-4. Thus, it can be finally determined ¹⁶⁴ HNFIQTI ¹⁷⁰ Is a central linear epitope that is recognized by McAb1B4G 2-4.

Based on the experimental analysis results, the inventor further analyzes the conservation of the epitope of McAb1B4G2-4, the spatial structure of the epitope identified by the McAb prepared in the application and the like by using relevant bioinformatics analysis software, and the specific cases are outlined below.

The McAb1B4G2-4 epitope obtained by the identification of the application is subjected to sequence alignment with 13 wild strains of typical ASFV at home and abroad screened from NCBI. The results are shown in FIG. 7 (FIG. 7A). It can be seen that: in 13 domestic and foreign ASFV strains, the epitope identified by McAb1B4G2-4 related to the application is highly conserved, which indicates that the epitope identified by the application has better universality in specific detection application and can be better used for detection application of different strains.

The visual analysis result (figure 7B) of the spatial structure of the p30 epitope shows that the McAb1B4G2-4 epitope is positioned on the outer side of the p30 protein, and the result shows that the McAb1B4G2-4 epitope is easier to be recognized by ASFV, so that a theoretical foundation can be laid for the application effect of the McAb1B4G2-4 epitope.

In general, the present application identifies the smallest linear epitope obtained ¹⁶⁴ HNFIQTI ¹⁷⁰ Has partial alpha spiral turning region and coil region, shows stronger antigen index, is highly conserved among different strains, and has better application guidance significance for the development of subsequent vaccines. Meanwhile, the related research results of the application also provide better reference and reference for the identification and development of other antigen epitopes, and have important technical significance for the development of final anti-ASFV vaccine.

Claims

1. The linear B cell epitope related to the African swine fever virus p30 protein is characterized in that the epitope is a polypeptide with 7 amino acid residues, the amino acid sequence of the polypeptide is shown as SEQ ID No.1, and the polypeptide specifically comprises: HNFIQTI.

2. Use of the linear B cell epitope related to the p30 protein of african swine fever virus according to claim 1 in the preparation of an african swine fever detection reagent.

3. Use of the linear B cell epitope related to the p30 protein of african swine fever virus according to claim 1 for preparing an african swine fever vaccine.

4. The gene sequence for encoding the African swine fever virus p30 protein is characterized by being shown as SEQ ID No. 2.