阅读说明：本技术 抗非洲猪瘟病毒p54蛋白单克隆抗体、制备方法及应用 (anti-African swine fever virus p54 protein monoclonal antibody, preparation method and application ) 是由 陈玉梅 王爱萍 蒋敏 赵建国 冯景 石海宁 赵孟孟 于 2020-12-30 设计创作，主要内容包括：本发明涉及抗非洲猪瘟病毒p54蛋白单克隆抗体、制备方法及应用。本发明设计并合成能够模拟保守性p54蛋白NTD的多肽,并将其与BSA偶联作为免疫原,通过免疫学方法免疫BALB/c小鼠制备获得抗ASFVp54蛋白单克隆抗体,该抗体能够特异性的识别并结合p54蛋白的N端区域,该识别区域(aa 1-29)区别于现有商品化单抗。本发明提供了上述抗体的重链可变区及轻链可变区基因序列,在此基础上,可以采用常规基因工程或蛋白质工程方法获得本发明的单克隆抗体。该单克隆抗体特异性强,灵敏度高,能与ASFV HLJ/18株病毒发生特异性反应,而与CSFV、PCV2、PRRSV、PEDV及PRV等猪源病毒不发生反应,为进行ASFV病原学和致病机理的研究及ASFV病原的临床检测研究奠定了基础。(The invention relates to an anti-African swine fever virus p54 protein monoclonal antibody, a preparation method and application. The invention designs and synthesizes polypeptide capable of simulating conservative p54 protein NTD, couples the polypeptide with BSA to be used as immunogen, and immunizes BALB/c mice by an immunological method to prepare and obtain the anti-ASFVp 54 protein monoclonal antibody, wherein the antibody can specifically recognize and combine with the N-terminal region of p54 protein, and the recognition region (aa 1-29) is different from the existing commercial monoclonal antibody. The invention provides the heavy chain variable region and light chain variable region gene sequences of the antibody, and on the basis, the monoclonal antibody can be obtained by adopting a conventional genetic engineering or protein engineering method. The monoclonal antibody has strong specificity and high sensitivity, can perform specific reaction with ASFV HLJ/18 strain virus, does not react with swine viruses such as CSFV, PCV2, PRRSV, PEDV, PRV and the like, and lays a foundation for the research on ASFV etiology and pathogenic mechanism and the clinical detection research on ASFV etiology.)

1. The African swine fever virus p54 protein monoclonal antibody is characterized in that a heavy chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.1-3, a CDR2 with an amino acid sequence shown as SEQ ID NO.2 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the variable region of the light chain of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown in SEQ ID NO.4, a CDR2 with an amino acid sequence shown in SEQ ID NO.5 and a CDR3 with an amino acid sequence shown in SEQ ID NO. 6.

2. The anti-African swine fever virus p54 protein monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 7; the amino acid sequence of the monoclonal antibody light chain variable region is shown in SEQ ID NO. 8.

3. The monoclonal antibody against p54 protein of African swine fever virus of claim 1 or 2, wherein the heavy chain constant region of the monoclonal antibody is lgG1 type, and the light chain constant region is Kappa type.

4. The monoclonal antibody against African swine fever virus p54 protein according to claim 1 or 2, wherein the monoclonal antibody specifically recognizes the sequence shown in SEQ ID No.11 in the N-terminal region of African swine fever virus p54 protein.

5. A nucleic acid molecule encoding the anti-african swine fever virus p54 protein monoclonal antibody of claim 1 or 2.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the gene encoding the heavy chain variable region of the anti-African swine fever virus p54 protein monoclonal antibody is shown in SEQ ID NO: 9 is shown in the figure; the gene nucleotide sequence of the variable region of the light chain of the monoclonal antibody for encoding the anti-African swine fever virus p54 protein is shown as SEQ ID NO: shown at 10.

7. A recombinant expression vector comprising the nucleic acid molecule of claim 5 or 6.

8. A host cell comprising the recombinant expression vector of claim 7 or having integrated into its genome the nucleic acid molecule of claim 5 or 6.

9. The method for preparing the African swine fever virus p54 protein monoclonal antibody according to claim 1 or 2, wherein the method comprises the following steps: culturing the host cell of claim 8 under suitable conditions.

10. The use of the African swine fever virus p54 protein monoclonal antibody of claim 1 or 2 in the preparation of an African swine fever virus detection reagent or kit.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an anti-African swine fever virus p54 protein monoclonal antibody, a preparation method and application.

Background

African Swine Fever Virus (ASFV) is a large and complex DNA virus that is the causative agent of African Swine Fever (ASF) and is the only known member of the genus Asfarviridae. ASF is a highly contagious hemorrhagic disease in domestic pigs, with morbidity and mortality rates as high as 100%. ASFV was first discovered in kenya in 1921 and has been around a century ago. Since the year 2007 to the state of georgia, ASFV has spread rapidly throughout several countries in africa, asia and europe, and outbreaks have occurred in these areas. At the same time, ASFV has multiple natural hosts and repositories, which further add to the potential threat of ASF. In view of the great risk and threat of such a cross-border animal disease, it has been placed in the world animal health Organization (OIE) terrestrial animal health code and is defined as an animal disease that must be reported to the OIE.

Due to the global economic importance of ASFV, since the end of the 1960 s, attempts have been made to develop an effective, safe vaccine to protect pigs against ASFV infection. However, due to the complex structure of ASFV and limited understanding of the function of viral proteins, a safe and effective vaccine against ASF has not been developed worldwide, and control of african swine fever is strictly dependent on animal quarantine. Therefore, the kit is of great importance for detection and prevention and control of ASFV.

ASFV has a large linear double-stranded DNA (dsDNA) genome of 170-194kb, encoding more than 150 proteins. Although as many as 68 structural proteins have been identified from virions, the function of most proteins is unclear. ASFV virions have a unique multilayer structure, and intracellular virions are composed of nucleosides (first layer), an inner nucleocapsid (second layer), an inner membrane (third layer), and a capsid (fourth layer). The extracellular virion has an additional outer envelope (fifth layer). Both intracellular and extracellular ASFV virions are infectious. The structural proteins constituting the virus particle are distributed in these multilayer structures and play various roles in viral infection. The identification of antigenic properties of viral structural proteins is essential for understanding the interaction between virus and host, and is crucial for improving serological diagnosis of ASFV and designing subunit vaccines or viral vector vaccines. At present, ASFV has 25 genotypes, and the genetic diversity and antigenic diversity of some genes are one of the main reasons for the lack of cross-protective vaccines against this lethal swine disease. The diversity of antigenic structural proteins may also have a tremendous impact on the reliable diagnosis of ASFV infection.

In order to deeply research the role of antigenic structural proteins in virus infection and realize accurate diagnosis of ASFV virus infection, more monoclonal antibodies capable of recognizing different structural proteins and different epitopes of ASFV are still needed in the field, and a new direction and a new idea are provided for preventing and treating ASF. The coding sequence of the African swine fever virus p54 protein has polymorphism among different strains, but the N-terminal region (NTD) of the African swine fever virus has relative conservation among various strains and has important function in the virus morphogenesis, invasion and other processes. However, no monoclonal antibody specific for NTD has been found in the prior art and reports. Therefore, the invention designs and synthesizes the polypeptide capable of simulating the protein NTD of p54, couples the polypeptide with BSA to be used as immunogen so as to obtain the monoclonal antibody for specifically recognizing the protein NTD of p54, provides a tool for further researching the characteristics of the antigenic structural protein p54 and the functions of different structural domains, and provides a new direction and a new thought for the prevention and control of ASF.

Disclosure of Invention

The invention aims to provide a monoclonal antibody against African swine fever virus p54 protein, which can specifically recognize the N-terminal region (aa 1-29) of the African swine fever virus p54 protein.

The second purpose of the invention is to provide a preparation method of the African swine fever virus p54 protein monoclonal antibody.

The third purpose of the invention is to provide the application of the African swine fever virus p54 protein monoclonal antibody.

In order to achieve the purpose, the invention adopts the following technical scheme:

the monoclonal antibody for resisting African swine fever virus p54 protein, wherein the heavy chain variable region of the monoclonal antibody comprises CDR1 with an amino acid sequence shown as SEQ ID NO.1-3, CDR2 with an amino acid sequence shown as SEQ ID NO.2 and CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the variable region of the light chain of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown in SEQ ID NO.4, a CDR2 with an amino acid sequence shown in SEQ ID NO.5 and a CDR3 with an amino acid sequence shown in SEQ ID NO. 6.

Specifically, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 7; the amino acid sequence of the monoclonal antibody light chain variable region is shown in SEQ ID NO. 8.

Specifically, the heavy chain constant region of the monoclonal antibody is of the lgG1 type, and the light chain constant region is of the Kappa type.

Specifically, the anti-African swine fever virus p54 protein monoclonal antibody can specifically recognize the African swine fever virus p54 protein; furthermore, the monoclonal antibody against the African swine fever virus p54 protein can specifically recognize the N-terminal region of the African swine fever virus p54 protein as shown in SEQ ID NO. 11; furthermore, the monoclonal antibody against the African swine fever virus p54 protein can specifically recognize a peptide fragment or an epitope with a DSEFFQPV sequence on the African swine fever virus p54 protein.

It is obvious to those skilled in the art that, on the basis of the heavy and light chain variable region amino acid sequences of the monoclonal antibody specifically disclosed in the present invention, one or more amino acid additions, deletions, substitutions and other modifications can be made by conventional protein engineering methods to obtain a conservative variant or a fragment thereof, while still maintaining specific binding with a peptide fragment or epitope having the DSEFFQPV sequence on the african swine fever virus p54 protein.

A nucleic acid molecule, wherein the nucleic acid molecule codes the anti-African swine fever virus p54 protein monoclonal antibody.

Specifically, the nucleotide sequence of the gene for coding the heavy chain variable region of the African swine fever virus p54 protein resistant monoclonal antibody is shown as SEQ ID NO: 9 is shown in the figure; the gene nucleotide sequence of the variable region of the light chain of the monoclonal antibody for encoding the anti-African swine fever virus p54 protein is shown as SEQ ID NO: shown at 10.

The antibody nucleic acid molecule can be obtained by using genetic engineering recombination technology or chemical synthesis method. It is obvious to those skilled in the art that the variant sequences of the heavy chain variable region nucleotide sequence and/or the light chain variable region nucleotide sequence obtained by mutation of the above-mentioned nucleic acid molecules provided by the present invention by one or more nucleotide additions, deletions, substitutions, modifications, etc., and the single-chain antibody or chimeric monoclonal antibody or modified monoclonal antibody or other forms of monoclonal antibody or antibody fragment consisting of the encoded amino acid sequences still retain the ability to specifically bind to the peptide fragment or epitope having the DSEFFQPV sequence on the african swine fever virus p54 protein.

A recombinant expression vector comprising the nucleic acid molecule described above.

Further, the recombinant expression vector is selected from prokaryotic or eukaryotic expression vectors; further, the recombinant expression vector is selected from the group consisting of bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, and other vectors.

A host cell comprising a recombinant expression vector as described above, or having a nucleic acid molecule as described above integrated into its genome.

Further, the expression system is a bacterial, yeast, filamentous fungus, mammalian cell, insect cell, plant cell or cell-free expression system.

A method for preparing a p54 protein monoclonal antibody of african swine fever virus, the method comprising the steps of: culturing the above host cell under appropriate conditions.

The African swine fever virus p54 protein monoclonal antibody is applied to preparation of an African swine fever virus detection reagent or kit.

The monoclonal antibody of the invention can specifically identify the African swine fever virus p54 protein and the African swine fever virus, and can be used for detecting the African swine fever virus p54 protein and also can be used for detecting the African swine fever virus.

Specifically, the detection reagent or the kit comprises a monoclonal antibody of the anti-African swine fever virus p54 protein.

The invention has the following beneficial effects:

the African swine fever virus p54 protein monoclonal antibody provided by the invention is prepared by designing and synthesizing a polypeptide capable of simulating a conserved p54 protein N-terminal region (NTD, aa 1-29), coupling the polypeptide with BSA to be used as an immunogen and immunizing a BALB/c mouse by an immunological method, and can specifically recognize and combine the NTD (aa 1-29) and a peptide segment of the p54 protein, wherein the recognition site (DSEFFQPV) is different from the existing commercial monoclonal antibody. In the process, we analyzed the conservation of p54 using bioinformatics, and by reviewing previous studies and extensive analysis, it was found that NTD is a key antigenic region within the p54 protein. Experiments prove that the antibody induced by the protein p54 mainly recognizes a linear epitope, and the anti-p 54 protein positive serum can specifically recognize NTD. In addition, p54 dimerization, which is required for mature virion formation, must occur through a unique cysteine residue located in the NTD. This suggests that NTD is a new target for the preparation of monoclonal antibodies (mabs) against the p54 protein and is also a key target for the study of AFSV morphogenesis. The monoclonal antibody prepared by taking the NTD polypeptide coupled BSA as the immunogen has strong specificity and high sensitivity, and the heavy chain type is as follows: lgG1, light chain type: kappa type, titer not less than 1: 1.024X 10⁶Has no cross reaction with other swine viruses such as Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), porcine pseudorabies virus (PRV) and the like, can be simultaneously used for a plurality of immunological detection means such as ELISA, IPMA and the like, and has good application prospect.

The invention also provides the variable region amino acid sequences and the nucleotide sequences of the heavy chain and the light chain of the African swine fever virus p54 protein monoclonal antibody, on the basis, the monoclonal antibody can be obtained by adopting a conventional genetic engineering or protein engineering method, and further, the active fragment or conservative variant can be obtained by adopting the modification of addition, deletion, substitution and the like of one or more amino acids, thereby laying the foundation for further improving the specificity and the affinity of the antibody.

Drawings

FIG. 1 is the serum titer of immunized mice;

in the figure, 21dpi is the mouse serum 21 days after immunization; 42dpi is the mouse serum 42 days after immunization.

FIG. 2 shows monoclonal antibody Western blotting identification;

in the figure, a hole 1 is a prokaryotic expression product pET28 a-P54; well 2 is the empty expression product of pET28 a;

FIG. 3 depicts monoclonal antibody-specific IPMA identification;

in the figure, A is pcDNA^TM3.1 IPMA identification of 293T cells transiently transfected with myc-HisA/ASFV P54; b is an untransfected 293T cell control

FIG. 4 shows the cross-reactivity of IPMA detection monoclonal antibody with other porcine viruses;

in the figure, A is ASFV HLJ/18 strain; b is PCV 2; c is CSFV; d is PRRSV; e is PEDV; f is PRV.

FIG. 5 shows p54 protein p54N₂₉B cell epitope indirect ELISA identification result graph;

in the figure, P1-P3 are overlapping polypeptides covering amino acids 1-29 of the P54 protein; NC is SMCC-BSA, and PC is polypeptide p54N as negative control₂₉As a positive control.

FIG. 6 is a truncated peptide library to determine the minimum mapping table;

in the figure, A is the reaction result of the N-terminal truncated polypeptide and the monoclonal antibody, and B is the reaction result of the C-terminal truncated polypeptide and the monoclonal antibody.

FIG. 7 shows p54 protein p54N₂₉The identification map of the key amino acid sequence of the B cell epitope of (1).

In the figure, E1 is²DSEFFQPV⁹An antigenic epitope; F5A, F6A, Q7A, P8A and V9A are polypeptides with alanine mutation at the corresponding sites of the antigen epitopes respectively.

FIG. 8 shows the application of the monoclonal antibody provided by the present invention in IPMA detection;

in the figure, A is a monoclonal antibody described in the present invention; b is ASFV pig positive serum as positive control; c is an anti-PCV 2Cap protein monoclonal antibody as a negative control.

FIG. 9 shows the application of the monoclonal antibody provided by the present invention in IFA detection;

in the figure, A is a monoclonal antibody described in the present invention; b is an anti-PCV 2Cap protein monoclonal antibody as a negative control.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the instruments and equipment involved in the following examples are conventional instruments and equipment unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.

EXAMPLE 1 selection and preparation of immunogens

The virus structural protein P54 is coded by E183L gene, is located in the inner membrane of the virus envelope, and the N end of P54 is relatively conserved among strains and contains only cysteine, thereby having important functions in the virus morphological formation, activity, invasion and other processes. In the previous experiment, a polypeptide is synthesized according to the NTD sequence of the p54 protein to simulate the NTD, the polypeptide has good reaction with the serum of an ASFV infected clinical recovery pig, and the p54 protein NTD region is proved to contain an antigenic determinant which can induce an organism to generate an antibody aiming at the NTD. Therefore, the p54 protein NTD is coupled with the carrier protein BSA as an immunogen in the invention. Specifically, the preparation of the immunogen comprises the following steps:

(1) a polypeptide which can simulate p54 protein NTD is synthesized by a solid phase synthesis polypeptide technology by taking a p54 protein sequence (GenBank: MH717102.1) of ASFV strain SY18 in the first ASF outbreak reported in China as reference, and is named as NTD, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.

(2) Polypeptide coupling carrier protein BSA (bovine serum albumin)

Coupling was performed using a water-soluble amino-mercapto crosslinker, Sulfo-SMCC. Sulfo-SMCC has two reactive groups, Sulfo-NHS ester and maleimide, and can react between primary amino and sulfhydryl. First, under the condition of pH7-9, Sulfo-SMCC reacts with the primary amine group of carrier protein BSA to form a stable amide bond, and the activated carrier protein BSA is obtained. Next, activated BSA was dialyzed against PBS (pH7.2-7.4) and the dialyzate was changed at least three times with 6-hour intervals. The dialyzed solution was collected and adjusted to a protein concentration of 5mg/ml with PBS. Finally, under the condition of pH 6.5-7.5, the activated BSA reacts with the sulfydryl of the polypeptide NTD to form a stable thioether bond, and a conjugate of the immunogenic carrier protein BSA and the polypeptide NTD is formed for antibody production.

EXAMPLE 2 preparation of monoclonal antibodies

1. Animal immunization

(1) Adding Freund's complete adjuvant into immunogen NTD-BSA, emulsifying and using for first immunization;

(2) immunizing 2 female BALB/c mice of 4-8 weeks old by a back subcutaneous multipoint injection method, wherein the immunization dose is 10 mu g/mouse;

(3) emulsifying the immune antigen with Freund's incomplete adjuvant at intervals of 3 weeks, and performing booster immunization on BALB/c mice by the same method and dosage;

(4) after three weeks, tail vein blood sampling is carried out to determine the specific antibody titer aiming at NTD, a mouse with higher titer (figure 1) is selected, and the BALB/c mouse is subjected to super-strong immunity by immunogen without adjuvant through a tail vein injection method 3-4 days before cell fusion, wherein the immunization dose is 20 mu g/mouse.

2. Cell fusion and monoclonal antibody preparation

The method of polyethylene glycol is adopted, and the spleen cells of the immunized mice and the mouse myeloma cells SP2/0 are mixed according to the cell number of 8: 1, and screening the fused cells by using HAT selective medium; 12 days after fusion, the positive hybridoma cells are primarily screened by an indirect ELISA method by taking polypeptide NTD as a coating antigen;

the indirect ELISA method comprises the following steps:

(1) diluting unconjugated NTD polypeptide with CBS solution to obtain coating solution with concentration of 2 μ g/mL, coating the ELISA plate with the coating solution, sealing at 4 deg.C overnight at 100 μ l/hole;

(2) diluting hybridoma supernatant (primary antibody) with 5% skimmed milk 2 times, sequentially adding into enzyme labeling plate at 50 μ l/well, and incubating at 37 deg.C for 30min, wherein the positive control is ASFV pig positive serum;

(3) discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(4) diluted HRP-labeled goat anti-mouse IgG (secondary antibody) was added to the reaction wells at 50 μ l/well. Incubating at 37 ℃ for 30 min;

(5) discarding the secondary antibody, washing with PBST, and patting dry;

(6) adding 100 mul of TMB color developing solution prepared in situ into each hole, and reacting for 15min in a dark room;

(7) add 50. mu.l of 2M H per well₂SO₄Terminating the reaction;

(8) microplate reader for reading OD of each well₄₅₀The value is obtained.

3. Subcloning of hybridoma cells by limiting dilution method

The positive hybridoma cells were diluted to about 1.5cells/ml with 1640/10 complete medium, 100. mu.l per well were added to a 96-well plate pre-plated with 100. mu.l feeder cells, and placed at 37 ℃ in 5% CO₂Culturing for 6-8 days in an incubator; further screening positive hybridoma cells by an indirect ELISA method; carrying out subcloning for 2-3 times until obtaining hybridoma cell strain which stably secretes anti-P54 protein monoclonal antibody, obtaining target hybridoma cell, carrying out expanded culture on the screened positive monoclonal antibody, and carrying out cell number according to 1-2 × 10⁶Freezing and storing in a tube.

4. Stability identification of monoclonal hybridoma cell strain

Continuously culturing the established monoclonal hybridoma cell strain for 3 months and repeatedly freezing and storing by liquid nitrogen for resuscitation so as to identify the stability of the hybridoma cell; the results show that the monoclonal hybridoma cell strain has good stability.

5. In vivo induced ascites method for preparing monoclonal antibody

Selecting female Balb/c mice, injecting 500 μ l sterilized paraffin into abdominal cavity, injecting obtained monoclonal hybridoma cell into abdominal cavity again after one week,the injection amount is 2X 10⁵After one week, ascites is extracted after the abdomen of the mouse is enlarged, the supernatant is centrifuged, and the ascites is purified by ammonium caprylate method.

EXAMPLE 3 purification and characterization of antibodies

1. The saturated ammonium sulfate precipitation method is used for purifying the antibody and the operation method is as follows:

(1) 5ml of monoclonal antibody ascites is taken, 5ml of PBS buffer solution is added, 2.5ml of saturated ammonium sulfate solution is added dropwise to obtain 20% ammonium sulfate solution, the ammonium sulfate solution is added and stirred, and after the mixture is fully and uniformly mixed, the mixture is kept stand for 30 min.

(2)8000r/min, centrifuging for 20min, and discarding the precipitate to remove fibrin.

(3) Adding 12.5ml saturated ammonium sulfate solution into the supernatant, mixing well, standing for 30 min.

(4)8000r/min, centrifuging for 20min, and discarding the supernatant.

(5) Dissolving the precipitate in 10ml PBS buffer solution, adding 5ml saturated ammonium sulfate solution to obtain 33% ammonium sulfate solution, mixing, and standing for 30 min.

(6)8000r/min, centrifuging for 20min, and discarding supernatant to remove albumin.

(7) And repeating the step 5, 2-3 times.

(8) The precipitate was dissolved in 5ml of PBS buffer, and the solution was placed in a dialysis bag, dialyzed against PBS buffer at 4 ℃ and changed 4 times.

(9)8000r/min, centrifuging for 20min, discarding precipitate to obtain supernatant as purified antibody, measuring antibody concentration, packaging, and storing at-20 deg.C.

2. Monoclonal antibody potency assay

The indirect ELISA assay was performed with reference to example 2, with a slight difference in primary antibody: diluting the purified monoclonal antibody with 5% skimmed milk at a ratio of 1:1000 by 2 times, sequentially adding into an enzyme label plate at 50 μ l/well, and incubating at 37 deg.C for 30min, wherein the positive control is ASFV pig positive serum; the other steps are carried out according to example 2, and the ELISA test result shows that the titer of the monoclonal antibody is 1: 1.024X 10⁶。

3. Subtype identification

The subtype of the Monoclonal Antibody is identified by a Mouse Monoclonal Antibody subtype identification Kit (Sigma), and the identification result shows that the Monoclonal Antibody belongs to IgG1 and the light chain type is Kappa type.

4. Identification of monoclonal antibody specificity

Western-blotting identification of monoclonal antibodies: subcloning the complete coding sequence (CDR) of the p54 gene (GenBank accession MH717102) into pET-28a plasmid, constructing a prokaryotic expression vector pET28a-p54, transforming the recombinant clone pET28a-p54 into Escherichia coli BL21(DE3) cells, and carrying out western blot detection by using a monoclonal antibody, wherein the result is shown in figure 2, the monoclonal antibody can specifically react with the prokaryotic expressed ASFV p54 protein and does not react with an empty vector;

diluting the monoclonal antibody according to a certain proportion, and respectively adding the diluted monoclonal antibody into eukaryotic expression vector pcDNA^TM3.1/myc-HisA/ASFV P54 transient transfected 293T cells, the result of the detection by IPMA is shown in FIG. 3, and the monoclonal antibody reacts specifically with the cell source ASFV P54 protein but not with the untransfected cells.

5. Identification of Cross-reactivity with porcine Virus

And simultaneously, diluting the monoclonal antibody ascites according to a certain proportion, respectively adding the diluted monoclonal antibody ascites into cells infected by PCV2, CSFV, PRRSV, PRV and PEDV, and determining whether the monoclonal antibody has cross reactivity with PCV2, CSFV, PRRSV, PRV and PEDV by using an IPMA detection method. The IPMA result is shown in figure 4, the monoclonal antibody only reacts with ASFV HLJ/18 strain, the reaction results with other viruses (PCV2, CSFV, PRRSV, PRV and PEDV) are negative, and the monoclonal antibody is proved to have good specificity with the ASFV reaction and no cross reaction with other common porcine viruses.

EXAMPLE 4 monoclonal antibody variable region Gene amplification and sequencing

1. Primer design

Designing a heavy chain variable region primer sequence according to the sequence characteristics of the mouse-derived monoclonal antibody:

P1：5’-cctggtGAGGAGTCTGGACCTG-3’；

P2：5’-AAATCGAGAAGCACA-3’。

design of light chain variable region primer sequence:

P3：5’-CCTGTCAGTCTTGGAGATCAA-3’；

P4：5’-TATCCGTTTGATTTCCAGCTT-3’。

PCR amplification

The variable region sequences of the monoclonal antibodies are respectively obtained by a molecular cloning technology and sent to Shanghai Biotechnology Limited company for sequencing.

The sequencing results were as follows: the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively shown by SEQ ID NO.10 and SEQ ID NO.11, and the deduced amino acid sequences of the corresponding heavy chain variable region and the light chain variable region are respectively shown by SEQ ID NO.8 and SEQ ID NO. 9. Further analyzing to obtain the amino acid sequences of the heavy chain variable region CDR of the monoclonal antibody as shown in SEQ ID NO. 1-3; the amino acid sequences of the light chain variable region CDR of the monoclonal antibody are respectively shown in SEQ ID NO. 4-6.

Example 5 identification of epitope recognized by monoclonal antibody

1. Design of overlapping polypeptides

3 segments of polypeptides are designed and synthesized by adopting a polypeptide scanning method, the entire NTD is covered, and 7 amino acids are overlapped between adjacent polypeptides. The amino acid sequence of the polypeptide fragment is shown in Table 1.

TABLE 1

Name of	Positioning	Amino acid sequence
			P1	1-15aa	MDSEFFQPVYPRHYG
P2	9-23aa	VYPRHYGECLSPVTT
			P3	18-29aa	LSPVTTPSFFST

2. Indirect ELISA screening for short peptides capable of binding to antibodies

Diluting the polypeptide with CBS buffer solution, coating an enzyme label plate, and carrying out temperature control at 37 ℃ for 2 h; washing the PBST for 2-3 times; sealing with 5% skimmed milk at 37 deg.C for 2 hr; the monoclonal antibody obtained in the embodiment 3 of the invention is used as a primary antibody to carry out indirect ELISA, and the incubation is carried out for 1h at 37 ℃; after washing the plate, adding HRP-labeled goat anti-mouse IgG (H + L), and incubating for 30min at 37 ℃; adding a TMB substrate color developing solution after washing the plate, stopping developing after 5min, and reading by using an enzyme-linked immunosorbent assay. The results show that the screened monoclonal antibody can react with the polypeptide P1(¹MDSEFFQPVYPRHYG¹⁵) Specific reactions occurred (fig. 5).

3. Identifying the antigen epitope recognized by the monoclonal antibody.

The result of systematically creating a truncated library from the two-terminal truncated positive polypeptide P1 is shown in FIG. 6, in which A is the reaction result of the N-terminal truncated polypeptide with the monoclonal antibody, B is the reaction result of the C-terminal truncated polypeptide with the monoclonal antibody, and it can be seen from FIG. 6 that the minimum epitope peptide required for epitope activity is "²DSEFFQPV⁹", after the smallest epitope peptide is identified, an alanine scanning library is designed to identify key residues in the epitope. The purity of all synthetic peptides was equal to or greater than 95%. Determining the reactivity of each peptide in the truncated peptide library and the alanine scanning peptide library with the monoclonal antibody by an ELISA method, and determining the epitope "²DSEFFQPV⁹"the key amino acid is⁶FQPV⁹(FIG. 7).

Example 6 application of monoclonal antibody in preparation of African swine fever virus detection reagent or kit

The embodiment provides an African swine fever virus detection reagent, which comprises the anti-African swine fever virus p54 protein monoclonal antibody provided by the invention. And the specificity of the detection reagent is verified by adopting IPMA and IFA detection methods respectively.

The African swine fever virus HLJ/18 strain infects primary alveolar macrophages of pigs, and is prepared into a monolayer cell reaction plate after methanol fixation treatment. The monolayer cell reaction plate infected by the ASFV HLJ/18 strain is provided by Harbin veterinary institute of Chinese academy of agricultural sciences.

The method for detecting the African swine fever infected cells by the IPMA method comprises the following specific steps:

(1) the monolayer cell reaction plate prepared above was taken out of the refrigerator, equilibrated to room temperature, and washed 1 time with PBST.

(2) The detection reagent (containing the African swine fever virus p54 protein-resistant monoclonal antibody provided by the invention) is used as a primary antibody, and 5% of skim milk 1:1000, and setting the positive control as ASFV pig positive serum and the negative control as anti-PCV 2Cap protein monoclonal antibody, wherein the sample adding amount is 50 μ l per well. Incubate at 37 ℃ for 30 min.

(3) PBST was washed 3 times with HRP-labeled goat anti-mouse IgG as secondary antibody. HRP-labeled goat anti-porcine IgG was added to the positive control wells as a secondary antibody. Incubate at 37 ℃ for 30 min.

(4) PBST was washed 3 times, added with AEC substrate color developing solution, developed for 10min at room temperature, and placed under an optical microscope to observe the dyeing result.

And (4) judging a result: typical reddish brown staining was observed for cells infected with African swine fever virus, and no reddish brown staining was observed for uninfected cells and negative controls. The results are shown in FIG. 8, which shows that the monoclonal antibody against African swine fever virus p54 protein can specifically detect ASFV infected cells.

IFA detection method can be performed with reference to IPMA detection method, with slightly different secondary antibodies, FITC-labeled goat anti-mouse secondary antibody was added at 1: 500 dilution and addition to corresponding wells and other steps were performed with reference to IPMA. No chromogenic substrate was required and the results were observed under a fluorescent microscope. And (4) judging a result: typical green fluorescence was observed for cells infected with African swine fever virus, and for uninfected cells and negative controls there was no fluorescence. The results are shown in FIG. 9, which shows that the monoclonal antibody against African swine fever virus p54 protein can specifically detect ASFV infected cells.

Meanwhile, the monoclonal antibody of the invention can also be used in an ELISA detection reagent or an ELISA detection kit, such as in example 2, or the monoclonal antibody of the invention is labeled with a label and then used in the ELISA detection reagent or the kit to detect ASFV or ASFV p54 protein; the monoclonal antibody of the invention can also be used as a primary antibody in Western blotting to detect ASFV or ASFV p54 protein, as in example 3; or the monoclonal antibody of the invention is used as a gold-labeled antibody or a capture antibody for immunochromatography test paper to detect ASFV or ASFV p54 protein.

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.

<110> Henan Zhongze bioengineering, Inc

<120> African swine fever virus p54 protein monoclonal antibody, preparation method and application

<160> 19

<170> PatentIn version 3.5

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<212> PRT

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<221> heavy chain variable region CDR1

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Gly Phe Ser Ile Thr Arg Asp Tyr Gly

1 5

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Ile Ser Tyr Ser Gly Ser Asn

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<221> heavy chain variable region CDR3

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Ala Leu Met Met

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Gln Ser Leu Val His Ser Asn Gly ASn Thr Tyr

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Asp Ser Ser Phe

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Ser Gln Ser Thr Leu Leu Pro Pro Thr

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Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu

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Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Arg Asp Tyr Gly

20 25 30

Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Ala

35 40 45

Tyr Ile Ser Tyr Ser Gly Ser Asn Ser Tyr Asn Pro Ser Leu Lys Ser

50 55 60

Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Glu

65 70 75 80

Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Leu

85 90 95

Met Met Thr Thr Cys Ala Ser Arg Phe

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Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln

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Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln

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Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Asn Asp Ser Ser Phe His

35 40 45

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

50 55 60

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile

65 70 75 80

Tyr Phe Cys Ser Gln Ser Thr Leu Leu Pro Pro Thr Phe Gly Gly Gly

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Thr Lys Leu Glu Ile Lys Arg Ile

100

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<212> DNA

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cctggtgagg agtctggacc tggcctggtg aaaccttctc agtctctgtc cctcacctgc 60

actgtcactg gcttctcaat cacccgtgat tatggctgga actggatccg gcagtttcca 120

ggaaataaac tggagtggat ggcctatata agttatagtg gtagtaatag ctataaccca 180

tctctcaaaa gtcgaatctc tatcactcga gacacatcca agaaccagtt cttcctggag 240

ttgaattctg tgactactga ggacacagcc acatattact gtgcccttat gatgacgacg 300

tgtgcttctc gattt 315

<210> 10

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<212> DNA

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<221> light chain variable region

<400> 10

cctgtcagtc ttggagatca agcctccatc tcttgcagat ctagtcagag ccttgtacac 60

agtaatggaa acacctattt agattggtac ctgcagaagc caggccagtc tccaaagctc 120

ctgatcaacg actcaagttt ccaccgattt tctggggtcc cagacaggtt cagtggcagt 180

ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggaatt 240

tatttctgct ctcaaagtac acttcttcct ccgacgttcg gtggaggcac caagctggaa 300

atcaaacgga ta 312

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<212> PRT

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<221> NTD

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Met Asp Ser Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly Glu

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Cys Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr

20 25

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<212> DNA

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<221> heavy chain variable region primer P1

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cctggtgagg agtctggacc tg 22

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<212> DNA

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<221> heavy chain variable region primer P2

<400> 13

aaatcgagaa gcaca 15

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<212> DNA

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<221> light chain variable region primer P3

<400> 14

cctgtcagtc ttggagatca a 21

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<212> DNA

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<221> light chain variable region primer P4

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tatccgtttg atttccagct t 21

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<212> PRT

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<221> P1 peptide

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Met Asp Ser Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly

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Val Tyr Pro Arg His Tyr Gly Glu Cys Leu Ser Pro Val Thr Thr

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Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr

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<221> epitope

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Asp Ser Glu Phe Phe Gln Pro Val

1 5