WO2016086554A1

WO2016086554A1 - General monoclonal antibody for african swine fever virus strains as well as preparation method therefor and application thereof

Info

Publication number: WO2016086554A1
Application number: PCT/CN2015/075183
Authority: WO
Inventors: 曹琛福; 花群义; 吕建强; 刘建利; 宗卉; 杨俊兴; 张彩虹; 孙洁; 唐金明; 廖立珊
Original assignee: 深圳出入境检验检疫局动植物检验检疫技术中心
Priority date: 2014-12-05
Filing date: 2015-03-27
Publication date: 2016-06-09
Also published as: CN104497137A; CN104497137B

Abstract

Disclosed are a general monoclonal antibody for African swine fever virus strains as well as a preparation method therefor. The method comprises: immunizing a mouse by using a purified p54 immune protein, fusing spleen cells of the immunized mouse with myeloma cells to prepare hybridoma cells, screening with three screening antigens so as to obtain the hybridoma cells that can stably secrete a monoclonal antibody, and then preparing the monoclonal antibody by using an in vivo or in vitro method, wherein the three screening antigens comprise a prokaryotically expressed p54 recombinant protein, an eukaryotically expressed p54 recombinant protein and an artificially synthesized polypeptide shown in Seq ID No.1.

Description

Universal monoclonal antibody of African swine fever virus strain, preparation method and application thereof

Technical field

The present application relates to the field of monoclonal antibodies, and in particular to a universal monoclonal antibody for use in African swine fever virus strains, and a preparation method and application of the monoclonal antibodies.

Background technique

African Swine Fever (ASF) is an acute, heat, and highly contagious disease caused by African Swine Fever Virus (ASFV). Its clinical symptoms are hyperthermia, skin congestion, Abortion, edema and organ bleeding, the mortality rate is as high as 100%. However, after a small number of strains infected domestic pigs, pigs developed subacute infection or recessive poisoning. The World Organisation for Animal Health (OIE) listed it as a Class A animal disease, which has been highly valued by countries all over the world. The disease has not yet occurred in China. China has established this disease as a type of animal infectious disease and has been studied as an animal disease. Sun Huaichang. Chinese Journal of Preventive Veterinary Medicine, 1999, 21(21): 117-119).

In recent years, African swine fever has spread from the African continent to Armenia, Ukraine, and Russia in the Eurasian continent. In particular, the epidemic situation in the Caucasus in recent years is severe and poses a great threat to China. The occurrence of African swine fever in Western Europe, South America and Eastern Europe has confirmed that the introduction of African swine fever will bring a devastating blow to the pig industry. China has included African swine fever in the 2011 National Animal Disease Surveillance Program, and identified Xinjiang, Inner Mongolia, Heilongjiang, Jilin, Liaoning, and Tibet as key monitoring provinces (Wang Hongyan, pig raising, 2011, 4:81-82).

At present, there is no effective vaccine. Therefore, it is successful to determine the infection status of pigs by rapidly diagnosing the antibody level of African swine fever, so as to formulate effective control and elimination measures, and timely and accurate diagnosis and detection and strict and effective blockade and culling are successful. An effective way to eliminate African swine fever. Diagnostic methods for African swine fever antibodies include agar diffusion test, ELISA, immunoblotting, immunofluorescence, and the like. At present, China's detection reagents for the diagnosis of African swine fever are mainly dependent on imports. There are few methods and reagents with independent intellectual property rights. In addition to the severe epidemic situation in neighboring countries, China urgently needs to establish its own testing methods and reagents.

Further, in the conventional antibody diagnostic method, since the live virus-infected cells are used to prepare the antigen, the detection procedure cannot be standardized in clinical use, and the risk of active poisoning is large, so it is not suitable for popularization and application. At present, the development of detection methods and reagents mostly uses genetic engineering methods to express antigens, mainly focusing on the research of genes such as p72, p54, p32, pp62, A104R, B602L, K205R, among which p72, p54 and the like are widely used. The preparation of monoclonal antibodies is also mainly concentrated on these structural proteins, and the commercial kits based on immunological methods thus established are currently only four in the world. However, due to the large regional differences between the African swine fever virus strains, the existing commercial kits are prone to occur during use. The detection of African swine fever caused by some strains.

Summary of the invention

The purpose of the present application is to provide a novel monoclonal antibody against the African swine fever virus strain, and a preparation method and application of the universal monoclonal antibody.

In order to achieve the above objectives, the present application adopts the following technical solutions:

In one aspect, the present invention discloses a method for preparing a universal monoclonal antibody of an African swine fever virus strain, which comprises immunizing a mouse with purified p54 immune protein, and merging the spleen cells of the immunized mouse with the mouse myeloma cells to prepare a hybrid. Tumor cells, then screened positive hybridoma cell lines using three screening antigens to obtain hybridoma cells stably secreting monoclonal antibodies against African swine fever virus, and then preparing African swine fever virus strains by in vivo or in vitro methods. a monoclonal antibody; wherein the three screening antigens include a prokaryotic expression p54 recombinant protein, a eukaryotic expression p54 recombinant protein, and a synthetic sequence of the sequence shown by Seq ID No. 1.

Seq ID No. 1: SSRKKKAAAA IEEE DIQFINPYQDQQWAEV.

It should be noted that the key of the present application is to screen hybridoma cell lines by using three screening antigens, wherein the artificially synthesized polypeptides represented by Seq ID No. 1 are specific to all existing African swine fever virus strains. Designed, the sequence is aligned in the NCBI database and shared by all African swine fever virus strains. Therefore, the monoclonal antibodies prepared by the hybridoma cell strains screened by them are versatile and can be used in different regions. Sources of African swine fever virus are tested to avoid missed detection.

It should also be noted that the key to the present application is to screen hybridoma cell lines using three screening antigens, especially to artificially design a synthetic polypeptide, so that the prepared monoclonal antibody has versatility; as for p54 immune protein Preparation and purification, preparation of prokaryotic expression p54 recombinant protein, preparation of eukaryotic expression p54 recombinant protein, preparation of specific conditions and steps for merging hybridoma cells, immunoscreening of hybridoma cells, etc. can be carried out by referring to existing conventional methods. It is not exhaustive here. After screening for obtaining hybridoma cells stably secreting monoclonal antibodies against African swine fever virus, the preparation method of the monoclonal antibodies in vivo or in vitro can also refer to existing conventional methods; for example, intraperitoneal injection of hybridoma cells into animals, such as In mice, ascites is collected, and the monoclonal antibody is isolated and purified; or, the monoclonal antibody produced by secretion is collected by in vitro culture of the hybridoma cells.

The hybridoma cells stably secreted against the monoclonal antibody against African swine fever virus are designated as hybridoma cell line SZCIQASFV1, and the microbial deposit number is: CCTCC No. C2014212; the classification is named: hybridoma cell hybridoma cell; preservation The time is: December 3, 2014; the depositary is: China's typical culture preservation center; the deposit address is: Wuhan Wuchang Wushan Wuhan University Depository Center, Hubei Province.

In the present application, the p54 immune protein is obtained by transferring the p54 gene fragment of the pMD18-T-p54 plasmid into the pET-52b(+) plasmid, and performing soluble expression in Escherichia coli DH5α, and then extracting and purifying the expressed protein, ie, P54 immune protein.

In the present application, the prokaryotic expression p54 recombinant protein is a purified p54 recombinant protein obtained by prokaryotic expression of pET-52b(+) plasmid in E. coli; eukaryotic expression of p54 recombinant protein is eukaryotic using pFastBac/NT-TOPO insect expression system The obtained p54 recombinant protein was expressed.

The other side of the application discloses a universal monoclonal antibody against the African swine fever virus strain which is secreted by the hybridoma cell line SZCIQASFV1 of accession number CCTCC No. C2014212.

It should be noted that, in fact, the universal monoclonal antibody of the African swine fever virus strain of the present application is obtained by the preparation method of the present application.

A further aspect of the present application discloses the use of a universal monoclonal antibody of the present application in the preparation of an African swine fever virus detection reagent or device.

In yet another aspect of the present application, an African swine fever virus enzyme-linked immunosorbent assay kit comprising the universal monoclonal antibody of the present application is specifically disclosed.

Based on the above studies, the present application also provides a hybridoma cell which secretes a universal monoclonal antibody of the African swine fever virus strain of the present application, and has a accession number of CCTCC No. C2014212.

It should be noted that the hybridoma cells of the universal monoclonal antibody secreting the African swine fever virus strain of the present application are, in fact, the stable secretory anti-Africa selected by the preparation method of the universal monoclonal antibody of the African swine fever virus strain. Hybridoma cells of a classical antibody to classical swine fever virus.

Due to the adoption of the above technical solutions, the beneficial effects of the present application are:

In the preparation method of the present application, hybridoma cells are screened by using three screening antigens, in particular, a polypeptide shared by all African swine fever virus strains is designed and synthesized as a screening antigen, so that the prepared monoclonal antibodies can be different. The detection of geographically sourced African swine fever virus strains greatly reduces the phenomenon of missed detection caused by geographical differences between strains, and improves the quality and efficiency of inspection and quarantine work.

DRAWINGS

Figure 1 is a PCR identification result of the recombinant expression plasmid pET-52b(+)3C/LIC-p54 in the examples of the present application;

Figure 2: shows the results of SDS-PAGE analysis of the induced expression product of pET-52b(+)3C/LIC-p54 recombinant plasmid in BL21(DE3) in the examples of the present application;

Figure 3 is a result of purification and immunoblot analysis of the induced expression product of pET-52b(+)3C/LIC-p54 recombinant plasmid in BL21(DE3) in the examples of the present application;

Figure 4 is a PCR identification result of the pFastBac-p54 recombinant plasmid and the Bacmid-p54 recombinant cosmid in the examples of the present application;

Figure 5 is a Western-blot identification result of the pFastBac/NT-TOPO insect expression system eukaryotic expression p54 recombinant protein in the examples of the present application;

Figure 6 is a diagram showing the results of analyzing the affinity of the universal monoclonal antibody of the African classical swine fever virus strain and the specific antigen epitope polypeptide A in the African swine fever virus p54 protein by using the ProteinOn XPR36 macromolecular interaction instrument in the examples of the present application. In the analysis chart of the output, ka:2.06E+051/Ms, kd:6.13E-041/s, KD: 2.97E-09M;

Figure 7 is a graph showing the results of an alignment analysis of the African swine fever virus p54 protein sequence in the examples of the present application.

detailed description

Due to the large regional differences among the strains of African swine fever virus, the existing commercial kits are prone to missed detection during use; there is a need for a strain of African swine fever virus that is good for different geographical sources. A monoclonal antibody that detects the effect. Therefore, the present application aims to develop a monoclonal antibody that can be used to establish a monoclonal antibody based on all known African swine fever virus strains that are currently prevalent worldwide. Immunological methods such as competitive ELISA methods for antibodies provide key materials and methods to solve the problem of missed detection of existing commercial kits, and reduce or do not rely on foreign detection reagents, so that China has independent detection capabilities and detection reagents to prevent African swine fever The epidemic was introduced to China.

Based on the above needs and purposes, the present application specifically designs and artificially synthesizes a polypeptide shared by all known African swine fever virus strains, ie, the sequence represented by Seq ID No. 1, for hybridoma cell screening, and The prokaryotic expression of p54 recombinant protein and eukaryotic expression p54 recombinant protein were screened, so that the finally prepared monoclonal antibody can detect all known African swine fever virus strains, that is, the universal monoclonal antibody of African swine fever virus strain. antibody. Among them, the artificially synthesized polypeptides as screening antigens are confirmed by NCBI database comparison. In theory, especially the monoclonal antibodies obtained by screening can detect all known African swine fever virus strains; the test in this application is also different. The strains from geographical origin were tested and the results were in agreement with the theory.

It should be noted that, in the present application, the polypeptide of the sequence shown in the synthetic Seq ID No. 1 is designed for the antigenic site "EDIQFINPYQ", and the sequence polypeptide represented by Seq ID No. 1 is designed to ensure the immune effect. It can be understood that, in the basic concept of the present application, in the case of ensuring the integrity of the antigenic site "EDIQFINPYQ", about 20 amino acids can be added or subtracted before and after the polypeptide sequence based on the sequence polypeptide represented by Seq ID No. 1. All of them belong to the protection scope of the present application; more amino acids may be added to the sequence polypeptide represented by Seq ID No. 1 of the present application without considering the cost of artificial synthesis.

Based on the above studies, the present application provides a universal monoclonal antibody against the African swine fever virus strain. And its applications. It can be understood that the monoclonal antibody of the present application has good versatility, can detect strains of different geographical origins, and greatly reduces the missed detection caused by the geographical difference of the strain; therefore, the kit can be widely used for preparing the kit. Inspection and quarantine work; the kit contains, in addition to the monoclonal antibody of the present application, of course, other conventional reagents for enzyme-linked immunoassay, which are not described herein.

The present application will be further described in detail below by way of specific embodiments and the accompanying drawings. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.

Example 1 Preparation of immune antigen

1. Amplification and purification of the target gene

A pair of primers were designed to amplify the target gene from the pMD18-T-p54 plasmid cloned with the African swine fever virus p54 gene. The pMD18-T-p54 plasmid was saved and provided by the Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau. This plasmid contained a 552 bp p54 gene having the sequence shown as Seq ID No. 10. The upstream primer of the primer was the sequence shown by Seq ID No. 2, and the downstream primer was the sequence shown by Seq ID No. 3.

Seq ID No. 2: 5'-CAGGGACCCGGTTATACTATTCTCATTGCTATCG-3’

Seq ID No. 3: 5'-GGCACCAGAGCGTTCAAGGAGTTTTCTAGGTC-3’

After the primers were synthesized by the primer synthesis company, PCR amplification was carried out using the pMD18-T-p54 plasmid as a template. The reaction conditions were 94 ° C for 1 min, 58 ° C for 1 min, 30 cycles, and 72 ° C for 5 min.

After the reaction, the product was amplified by 1.5% agarose gel electrophoresis, and the purified target DNA was recovered by a gel recovery kit to obtain a target gene.

2. Construction of recombinant plasmid

Add T4 DNA polymerase to the target gene obtained in the above step, incubate at 22 °C for 30 min, incubate at 75 °C for 20 min, add 3C/LIC vector, incubate for 5 min at 22 °C, incubate for 5 min at 22 °C after adding EDTA, construct pET-52b(+)3C /LIC-p54 recombinant plasmid.

3. Transformation and identification of recombinant plasmid

The pET-52b(+)3C/LIC-p54 recombinant plasmid obtained in the above step was transferred into Escherichia coli DH5α, and inoculated on LB nutrient agar containing 100 mg/L ampicillin, and cultured at 37 ° C for 12 h, and a single colony was picked up. After incubating in LB liquid medium of 50 mg/L ampicillin for 12 hours at 37 ° C, the plasmid was extracted, and PCR identification and sequencing were performed to detect the integrity of the target gene.

PCR identification was performed using two pairs of primers, the first pair was the primer pair of Seq ID No. 2 and Seq ID No. 3, and the second pair was pET-52b(+)3C/LIC plasmid. Comes with a T7 primer pair. The results of gel electrophoresis identified by PCR are shown in Figure 1. In the figure, the M lane is a DNA marker. The lane 1 is the electrophoresis result of the first pair of primers PCR amplification, and the lane 2 is the electrophoresis result of the second pair of primers PCR amplification; it can be seen that the pET-52b(+)3C/LIC-p54 recombinant plasmid of this example is successfully constructed, and contains A target gene of about 462 bp is in line with theoretical expectations. The sequencing results showed that the recombinant plasmid contained 462 bp of p54 gene, and its sequence was consistent with the sequence shown by Seq ID No. 10. This further confirmed that the pET-52b(+)3C/LIC-p54 recombinant plasmid constructed in this example contains what we need. P54 gene.

It should be noted that the front end of the p54 gene contains two promoters, and in this case, only one of the promoters was cloned in prokaryotic expression, that is, the nucleotide sequence of the signal peptide of the front end of the p54 gene of 90 bp was not cloned, so A pair of amplified target genes, Seq ID No. 2 and Seq ID No. 3, showed a primer size of 462 bp, and the gene also contained the entire gene sequence of the p54 protein, but did not contain a signal peptide of 90 bp in front. Nucleotide sequence; for this example, does not affect prokaryotic expression of the p54 protein.

4. Induced expression of recombinant plasmid in BL21(DE3)

BL21(DE3) bacterial competent cells were prepared, and the recombinant plasmid pET-52b(+)3C/LIC-p54 was transformed into BL21(DE3) cells, and then cultured on LB plate containing 100 mg/L ampicillin to pick up a single colony. Inoculated in 2 mL of LB medium containing 100 mg/L ampicillin, and cultured at 37 ° C, 200 r / min shaking shaker to an OD600 value of about 0.6. IPTG was added to a final concentration of 2 mM, and the bacterial solution was collected after 2 hours of culture. The culture solution was centrifuged at 4 ° C for 15 min, the supernatant was removed, and the cells were collected. The pellet was resuspended in PBS, centrifuged at 10,000 g for 15 min at 4 ° C, and the supernatant was removed. The cells were resuspended in a sterilizing buffer, sonicated on ice, centrifuged at 4 ° C for 15 min, and the supernatant was collected. The expression product was stained with Coomassie brilliant blue by denaturing discontinuous SDS-PAGE, and the results were observed.

The results are shown in Figure 2. In the figure, the M lane is the protein molecular mass standard; the lanes 1-5 are 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM IPTG induces pET-52b(+)3C/LIC-p54 two hours later. The results of the analysis of the bacterial lysate supernatant; the 6 lanes were the results of the analysis of the bacterial lysate supernatant after two hours of pET-52b(+)3C/LIC-p54 induction without IPTG; the lanes of the bacteria were induced by 1 mM IPTG for two hours after BL21(DE3) The results of the analysis of the lysed supernatant; the results of the analysis of the bacterial lysate supernatant after pET-52b(+)3C/LIC-p541h, 2h, 3h, 4h, and 5h were induced by 1 mM IPTG in lanes 8-12; 13 lanes were without IPTG. The results of the analysis of the bacterial lysate supernatant after induction of pET-52b(+)3C/LIC-p54 two hours; the 14 lanes were 1 mM IPTG induced BL21 (DE3) two hours after the bacterial lysis supernatant. The results showed that the pET-52b(+)3C/LIC-p54 recombinant plasmid constructed in this example stably expressed the desired p54 recombinant protein.

5. Identification of expression products

The bacterial solution cultured in the above step was centrifuged at 4 ° C for 15 min, and the supernatant was removed to collect the cells. The pellet was resuspended in PBS, centrifuged at 10,000 g for 15 min at 4 ° C, and the supernatant was removed. The cells were resuspended in a sterilizing buffer, sonicated on ice, centrifuged at 4 ° C for 15 min, and the supernatant was collected. The expression product was subjected to immunoblot analysis using African porcine standard positive serum to detect the antigenicity of the protein of interest. The results of immunoblot analysis are shown in Figure 3. In the figure, M is the molecular weight standard of the protein, 1 is the sample washing solution, 2 is the purified sample of the target protein, 3 is the sample lysate, 4 is the western blot analysis of the target protein; the result shows that the recombinant protein obtained in this example is indeed p54 recombination. protein.

6. Purification of expression products

The supernatant of the sonication on ice was purified by Ni column affinity chromatography, and the purified product, that is, the immunogen for p54, which is required in this example, was purified.

Example 2 Preparation of screening antigen

In this example, three kinds of screening antigens were used to screen hybridoma cells. One is the prokaryotic expression of p54 recombinant protein as the immunizing antigen in Example 1, and the p54 immunoprotein purified by nickel column is used as screening antigen. The second is artificial synthesis. The method of synthesizing the polypeptide represented by Seq ID No. 1; the third is to use the pFastBac/NT-TOPO insect expression system to express the p54 recombinant protein, and the supernatant of the infected recombinant baculovirus cells is used as a screening antigen.

The synthetic polypeptide represented by Seq ID No. 1 is based on the p54 protein sequence of all African swine fever viruses which have been disclosed in this example, and the polypeptide sequences shared by all African swine fever viruses, therefore, As a screening antigen, it is theoretically possible to screen out all monoclonal antibodies against African swine fever virus, and the results are shown in Fig. 7.

The specific preparation of the antigen is as follows:

Seq ID No. 1: SSRKKKAAAA IEEEDIQFINPYQDQQWAEV

1. Synthesis of primers

Three sets of primers were synthesized according to the pFastBac/NT-TOPO insect expression system. The first group was the p54 gene amplification primer pair, the upstream primer NT-TOPO-ASFV54F was the sequence shown by Seq ID No. 4, and the downstream primer NT-TOPO-ASFV54R was The sequence shown by Seq ID No. 5; the second set is a primer pair for PCR identification of the pFastBac/NT-TOPO-p54 recombinant plasmid, the upstream primer Polyhedrin forward primer is the sequence shown by Seq ID No. 6, and the downstream primer SV40polyA reverse The primer is the sequence shown by Seq ID No. 7. The third group is the primer pair for PCR identification of Bacmid-p54 recombinant cosmid. The upstream primer pUC/M13Forward is the sequence shown by Seq ID No. 8, and the downstream primer pUC/M13Reverse The sequence shown in Seq ID No. 9.

Seq ID No. 4: 5'-ATGGATTCTGAATTTTTTCAACC-3’

Seq ID No. 5: 5'-TTACAAGGAGTTTTCTAGGTCT-3’

Seq ID No. 6: 5'-AAATG ATAAC CATCT CGC-3’

Seq ID No. 7: 5'-GGTAT GGCTG ATTAT GATC-3’

Seq ID No. 8: 5'-CCCAG TCACG ACGTT GTAAA ACG-3’

Seq ID No. 9: 5'-AGCGG ATAAC AATTT CACAC AGG-3’

2. Construction of pFastBac-p54 recombinant transposon vector

The same pMD18T-p54 recombinant plasmid as in Example 1 was used as a PCR template, and the p54 gene was amplified using the first set of primers, that is, the p54 gene amplification primer, and the PCR product was recovered using the PureLink HiPure Mini Plasmid Purification Kit to obtain the target gene. . 4 μL of the target gene was ligated to 1 μL of pFastBac/NT-TOPO vector, and then DH5α competent cells were transformed.

Positive cloned bacteria were picked and identified by PCR using the above two primers. First, PCR was performed on the NT-TOPO-ASFV54F and NT-TOPO-ASFV54R primers, which are the first set of primers. The second is the use of primer pairs NT-TOPO-ASFV54F and SV40polyA reverse primer. The results showed that the first set of primers amplified a fragment of about 552 bp, which was consistent with the expected 552 bp p54 gene. The second set of primers amplified the fragment size of about 758 bp, indicating that the target gene has been ligated to the appropriate position of the vector, and the direction of the connection. Correctly, the pFastBac-p54 recombinant plasmid was obtained. The results of partial gel electrophoresis of PCR amplification are shown in Figure A, where M is DNA Marker,

lanes

1 and 2 are electrophoresis results of the second set of primer amplification products, and 1 is a negative colony plasmid amplification product. 2 is a positive colony plasmid amplification product.

3. Construction of recombinant cosmid Bacmid-p54

10 μL of the recombinant plasmid pFastBac-p54 was transformed into E. coli DH 10Bac competent cells. After the transformation, 900 μL of LB medium was added to the EP tube, and cultured at 37 ° C for 4 hours at 225 rpm. The LB medium was diluted at a concentration of 1:10, 1:100, and 1:1000 at room temperature, and coated with 50 μg/mL kanamycin (hereinafter referred to as K+) and 7 μg/mL gentamicin. (hereinafter referred to as G+), 10 μg/mL tetracycline (hereinafter referred to as Tet+), 100 μg/mL of X-Gal, and 40 μg/mL of IPTG on LB screening plates, and cultured at 37 ° C for 48 hours. At least 10 white spots were picked, inoculated onto the screening plate of the above composition, and colonies were streaked and cultured overnight at 37 ° C to confirm whether it was still white spots. The colonies which were finally determined to be white spots were re-inoculated into 20 mL of LB medium containing K+, G+ and Tet+, and cultured overnight at 37 ° C in a shaker at 225 rpm. At the same time, Bacmid-CAT and Bacmid-HTA were subjected to the same operation, and were set as positive and negative controls, respectively. One part of the bacterial solution was taken out, and Bacmid was extracted using a large plasmid extraction kit PureLink HiPure Plasmid DNA Miniprep Kit, and the remaining bacterial liquid was stored at 4 °C. PCR was performed using two sets of primers: one was to use the first set of primers to amplify the target gene primers for PCR of NT-TOPO-ASFV54F and NT-TOPO-ASFV54R; the other was to use the third set of primers, ie primer pair pUC /M13Forward and pUC/M13Reverse perform PCR. A partial gel electrophoresis pattern of the PCR amplification product is shown in Figure B, where M is DNA Marker, and 3 is the amplification product of the first set of primers, namely primer pair NT-TOPO-ASFV54F and NT-TOPO-ASFV54R, 4 is the amplification product of the third set of primers, ie, primer pair pUC/M13Forward and pUC/M13Reverse, and 5 is a negative control. Result The first set of primers amplified a fragment of about 552 bp, which was consistent with the expected 552 bp p54 gene; the third set of primers amplified a fragment of about 2988 bp, indicating that the p54 gene has been ligated to the baculovirus vector. After the identification was completed, the remaining bacterial solution stored at 4 ° C was prepared into glycerol bacteria and frozen at -80 °C.

4. Acquisition of recombinant baculovirus AcNPV-p54

press

The IIReagent Transfection Kit instructions were used to lipidate Bacmid-p54, Bacmid-CAT, Bacmid-HTA. Sf9 insect cells were transfected after lipidation, while normal Sf9 insect cells not transfected with Bacmid DNA were used as blank controls. The cells were cultured in a 28 ° C incubator for approximately 72 h, during which time the cytopathic condition was observed. After about 36 hours, the cells showed lesions, and 80% of the cells showed lesions around 72 hours, and the cell culture supernatant was collected, which contained the recombinant baculovirus. The supernatant was re-infected with normal cells, and the cytopathic condition was observed. 80% of the cells developed lesions in about 72 hours, and the supernatant and cells were collected. The cells infected with the recombinant baculovirus were sonicated and the supernatant was extracted as a screening antigen.

The screening antigen was identified by Western-blot. The results are shown in Figure 5. In the figure, M is the protein marker, 1 is the normal sf9 cell protein, and 2 is the p54 recombinant protein. The results show that the supernatant contains the expected eukaryotic expression. P54 recombinant protein

Example 3 Preparation of Monoclonal Antibodies

Animal immunity

The purified p54 immunoprotein prepared in Example 1 was mixed with an equal amount of Freund's complete adjuvant and emulsified, and 7-week-old BALB/c female mice, 0.2 μg each, were injected subcutaneously. After 14 days, an equal amount of Freund's incomplete adjuvant was mixed with p54 protein and emulsified, and subcutaneous injection was performed. After 21 days, an equal amount of Freund's incomplete adjuvant was mixed with p54 protein and emulsified, and subcutaneous injection was performed. A booster injection without any adjuvant was performed 3 days prior to the planned and SP2/0 cell fusion.

2. Establishment of positive hybridoma cell lines

The immunized mice with high titer were mixed with SP2/0 myeloma cells in a ratio of 10:1, and hybridoma cells were prepared by conventional PEG fusion method. The supernatant of each well of hybridoma cells was detected by indirect ELISA using the screening antigen prepared in Example 2, and Escherichia coli and Sf9 cell proteins were used as negative controls for screening antigens.

The wells satisfying the indirect ELISA results of the three screening antigens in Example 2 were taken, subcloned by the limiting dilution method, the hybridoma cells were diluted 5 times and the monoclonal antibody test was repeated 5 times, and the last 3 times. In the antibody test, all the positive rate holes were 100%, and a hybridoma cell strain stably secreting monoclonal antibodies was obtained.

3. Preparation of monoclonal antibodies

Take 10 weeks old BALB/c female rats, intraperitoneal injection of liquid paraffin, each 0.5mL, 1 week later to the abdominal cavity 5 x 106 positive hybridoma cells were injected. After 7-10d, the abdomen of the mice was swollen obviously. The ascites was collected and centrifuged at 2000r/min for 5min. The supernatant was ascites containing anti-African swine fever p54 protein monoclonal antibody, which is the universal strain of African swine fever virus. Monoclonal antibodies were stored at -80 °C until use.

4. Identification of monoclonal antibody subclasses

The reagent in the Sigma monoclonal antibody subclass identification kit will be returned to room temperature. In the small test tube containing the test paper card, 500 μL of the buffer solution is added first, then 500 μL of the collected mouse ascites is added, and the small test tube is gently shaken to make the test paper. The card is fully submerged. Add 1 mL of anti-mouse antibody colloidal gold conjugate, keep the word side of the test paper card immersed in the liquid, and gently shake the small tube at room temperature until a small spot appears. It was identified that the monoclonal antibody secreted by this hybridoma cell was an IgG1 subclass, which was consistent with expectations.

5. Monoclonal antibody specific identification

Using the whole virus protein coated ELISA plate of classical swine fever virus, swine blue virus, foot-and-mouth disease virus, swine influenza virus and swine rabies virus as the detection antigen, the common monoclonal antibody of African swine fever virus strain prepared by this example was used. The antibody was detected as an antibody by indirect ELISA. The results showed that the universal monoclonal antibody of the African swine fever virus strain prepared in this example did not cross-react with the above-mentioned common swine pathogens and had good specificity.

6. Monoclonal antibody affinity analysis

Using the ProteinOn XPR36 macromolecular interaction system, the affinity between the universal monoclonal antibody of the African swine fever virus strain prepared in this example and the specific antigen epitope polypeptide A in the African swine fever virus p54 protein was investigated to determine the antigen-antibody relationship. The degree of dissociation is combined to determine the stability of the antigen-antibody complex. First, the chip was activated, and the universal monoclonal antibody of the African swine fever virus strain prepared in this example was chemically bonded to the chip, and then the chip was deactivated and tested for the binding of the monoclonal antibody. The formulated different concentrations of polypeptide A were used as the liquid phase, and the solid phase chip combined with the monoclonal antibody was passed at a flow rate of 25 μL/min. When the antigen-antibody binding peaked, the antigen-antibody complex was dissociated and tested for dissociation. rate.

The results are shown in Fig. 6. In the figure, the abscissa 0s-150s is the mutual binding effect of different concentrations of polypeptide A and monoclonal antibody, 150s-500s is the dissociation of the antigen-antibody complex; 1 is the polypeptide A with a concentration of 150nM, 2 is a polypeptide having a concentration of 75 nM, A is a polypeptide having a concentration of 37.5 nM, 4 is a polypeptide having a concentration of 18.75 nM, 5 is a polypeptide having a concentration of 9.375 nM, and 6 is a PBST buffer.

The results showed that the dissociation rate constant (Kd) of the universal monoclonal antibody of the African swine fever virus strain prepared in this example and the specific antigen epitope polypeptide A of the African swine fever virus p54 protein at room temperature was 6.13×10-41/s. The equilibrium binding constant (KD) is 2.97×10-9M, and the binding rate constant (Ka) is 2.06×1051/Ms; indicating the universal monoclonal antibody and specific epitope polypeptide of the African swine fever virus strain prepared in this example. A has a good combination and strong affinity, which is suitable for detection and diagnosis.

Example 4 Application of Monoclonal Antibodies

1. Materials and methods

1.1 serum and c-ELISA kit

African porcine standard positive sera were purchased from the IAH Pirbright laboratory in the United Kingdom; 153 negative sera were collected from multiple farms in the country; c-ELISA commercial kits were purchased from INGENASA, Spain.

1.2 Preparation of coated antigen

The purified antigen prepared in Example 1, i.e., p54 immunoprotein, was used to determine the protein concentration for coating the ELISA plate.

1.3 Preparation of monoclonal antibodies

The universal monoclonal antibody of the African swine fever virus strain prepared in Example 3 was labeled with horseradish peroxidase (HRP) and stored at -80 °C until use.

1.4 competition ELISA procedures

Coating: The P54 immune protein antigen was diluted to 2.5 μg/mL with a coating solution, and added to a 96-well microtiter plate, 100 μL per well, placed in a refrigerator at 4 ° C overnight, or at 37 ° C for 1 h.

Washing the plate: Remove the enzyme-labeled plate and discard the liquid. Add 200 μL of the washing solution to each well and wash the plate for three times. The last time it was inverted and patted dry.

Blocking: 100 μL of blocking solution was added to each well, and reacted at 37 ° C for 1 h to wash the plate.

Loading: Sample and weak positive control, positive control, negative control were diluted 1:5 with dilution, mixed, added to ELISA plate, 50 μL per well; react at 37 ° C for 1 h, wash the plate.

Add the enzyme-labeled monoclonal antibody conjugate in 1.3: Add 50 μL IgG-HRP per well and mix gently. The plate was placed at 37 ° C for 30 min, the reaction was completed, and the plate was washed.

Add substrate solution: Add 100 μL of substrate solution to each well and apply for 10 min at 37 °C.

The reaction was terminated: the reaction plate was taken out, and 50 μL of the stop solution was quickly added to each well, and the OD value was measured within 30 minutes.

Absorbance value: The absorbance was measured at 450 nm using a microplate reader.

1.5 Optimization of experimental conditions for competitive ELISA

1.5.1 screening of antigen coating concentration

According to the indirect ELISA experimental procedure, the purified p54 was coated in a concentration gradient: 5, 2.5, 1.25, 0.625 μg/mL, 50 μL/well, and coated at 4 ° C overnight. Enzyme-labeled monoclonal antibody 06-HRP vertical dilution: 25 × -3200 ×, 0.5 ° at 37 ° C, color development for 3 min, add 0.5 M sulfuric acid 50 μL / hole to stop color development, select OD1.5 or so, and consider the package The optimal coating concentration is selected by the cost and the number of enzyme-labeled monoclonal antibodies.

1.5.2 Screening of working concentration of enzyme-labeled monoclonal antibody

According to the above 1.4 competition ELISA experimental procedure, 2.5 μg/mL of p54 purified protein was coated with the enzyme labeling plate, and the standard positive serum to be tested was diluted by 40×, 80×, 160×, 320× longitudinal ratio, and repeated. The standard negative serum to be tested was diluted as 40× as a control, and duplicate wells were also made. The competition 06-HRP mAb was added horizontally and diluted by gradient: 100 x, 200 x, 400 x, 800 x. The OD value was measured, the inhibition rate was calculated, and the dilution ratio of the monoclonal antibody with the highest inhibition rate was taken as the optimal working concentration of the enzyme monoclonal antibody.

1.6 Competition ELISA method results determination threshold determination

In order to determine the critical value of the negative positivity test by competitive ELISA test method, 20 negative sera were used as the test serum, and the 20 sera were tested as negative serum by the African porcine antibody ELISA test kit of INGENASA, Spain. The optimized competitive ELISA method was tested in a test procedure and the OD value of each serum was determined. The blocking rate of the 20 sera, ie the PI value, was calculated according to the following formula:

PI (%) = (1 - sample serum mean OD value ÷ negative serum control mean OD value) × 100%

The average PI value of 20 sera and its standard deviation S were calculated. The value of the average PI value plus the standard deviation S of 3 times is used as a positive result to determine the critical value; the average PI value plus the value of the standard deviation S of 2 times is used as a negative result determination threshold value; and between the two, it is determined as a suspicious sample.

1.7 Competitive ELISA method for the detection of field samples

Take 107 serum samples from different countries, including 53 positive serum samples infected with different strains and 54 negative serum samples, which are tested according to the above optimized operating procedures and criteria, and are in compliance with commercial test kits. Sex comparison.

2 results

2.1 Screening of antigen concentration

According to the indirect ELISA experimental procedure, the purified p54 was coated in a concentration gradient: 5, 2.5, 1.25, 0.625 μg/mL, 50 μL/well, and coated at 4 ° C overnight. The above-mentioned enzyme standard monoclonal antibody was diluted in a vertical ratio: 25×-3200×, 0.5 h at 37° C., color development for 3 min, and 50 μL/well of 0.5 M sulfuric acid was added to terminate the color development. The results are shown in Table 1.

Table 1 antigen concentration screening results

The protein coating concentration at the OD450nm was about 1.5 and the concentration of the monoclonal antibody was measured. The results of the monoclonal antibody titer were determined, and the experimental combination with less antigen and antibody dosage was considered as the optimal working condition to determine the optimal coating concentration of the protein. 2.5 μg/mL.

2.2 Screening of the best enzyme monoclonal antibody working concentration

According to the above 1.4 competition ELISA experimental procedure, 2.5 μg/mL of p54 purified protein was coated with the enzyme labeling plate, and the standard positive serum to be tested was diluted by 40×, 80×, 160×, 320× longitudinal ratio, and repeated. The standard negative serum to be tested was diluted as 40× as a control, and duplicate wells were also made. The above competitive enzyme monoclonal antibody was added laterally and diluted by gradient: 100×, 200×, 400×, 800×. The OD value was measured and the results are shown in Table 2.

Table 2 Screening results of the best enzyme monoclonal antibody working concentration

The inhibition rate was calculated, and the dilution ratio of the monoclonal antibody with the highest inhibition rate was taken as the optimal working concentration of the enzyme monoclonal antibody. When the enzyme-labeled monoclonal antibody was diluted 100-fold, the positive serum had the highest inhibition rate, so the dilution factor of the enzyme-labeled monoclonal antibody was selected as 100 times as the optimal enzyme-labeled monoclonal antibody working concentration.

2.3 Determination of Critical Value and Judging Criteria of Competitive ELISA Method

In this experiment, 20 local negative sera were used as the test serum, and the 20 sera were tested as negative sera by the commercial test kit, and tested according to the optimized competitive ELISA method test procedure described above, and then the OD value of each serum was determined. The average PI value of 20 sera and its standard deviation S were calculated. The value of the average PI value plus the standard deviation S of 3 times is used as a positive result to determine the critical value; the average PI value plus the value of the standard deviation S of 2 times is used as a negative result to determine the critical value; and between the two, the suspicious sample is determined. The specific results are shown in Table 3.

Table 3 Critical value calculation results

2.4 Competition ELISA method field trial

Take 107 serum samples from different countries, including 53 positive samples infected with different strains Serum samples and 54 negative serum samples were tested according to the optimized operating procedures and criteria described above, and compared with commercial test kits for compliance. The results showed that 53 positive serum samples detected by the method established in this experiment were all positive, and 54 negative serum samples were all negative. Of the 53 positive serum samples detected by the African swine fever antibody ELISA kit from INGENASA, Spain, 51 were detected.

It can be seen that the competitive ELISA method established by the universal monoclonal antibody of the African swine fever virus strain prepared in this example can effectively detect the African swine fever virus strains from different geographical sources, and the detection rate is 100%, and there is no missed detection. . In this case, the universal monoclonal antibody of African swine fever virus strain and its competitive ELISA method can effectively replace the imported kit, establish a detection reagent in line with international trade and an animal disease monitoring method suitable for China's national conditions.

The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. It will be apparent to those skilled in the art that the present invention can be made in the form of the present invention without departing from the scope of the present invention.

Claims

The invention relates to a method for preparing a universal monoclonal antibody of an African swine fever virus strain, which comprises: immunizing a mouse with purified p54 immune protein, and merging the spleen cells of the immunized mouse with the mouse myeloma cells to prepare a hybridoma cell. Then, the positive hybridoma cell lines are screened by using three screening antigens to obtain hybridoma cells stably secreting monoclonal antibodies against African swine fever virus, and then the general monoclonal antibody of African swine fever virus strain is prepared by in vivo or in vitro methods. The three screening antigens include a prokaryotic expression p54 recombinant protein, a eukaryotic expression p54 recombinant protein, and a synthetic sequence of the sequence shown by Seq ID No. 1.

Seq ID No. 1: SSRKKKAAAA IEEE DIQFINPYQDQQWAEV.
The preparation method according to claim 1, wherein the p54 immunoprotein is obtained by transferring the p54 gene fragment of the pMD18-T-p54 plasmid into the pET-52b(+) plasmid and in Escherichia coli DH5α. Soluble expression was carried out, and then the purified expressed protein, p54 immunoprotein, was extracted.
The preparation method according to claim 1, wherein the prokaryotic expression p54 recombinant protein is a purified p54 recombinant protein obtained by prokaryotic expression of pET-52b(+) plasmid in Escherichia coli; the eukaryotic expression p54 The recombinant protein is a p54 recombinant protein obtained by eukaryotic expression using the pFastBac/NT-TOPO insect expression system.
A universal monoclonal antibody against an African swine fever virus strain, characterized in that the universal monoclonal antibody is secreted by the hybridoma cell line SZCIQASFV1 of the accession number CCTCC No. C2014212.
The universal monoclonal antibody according to claim 4, wherein the universal monoclonal antibody is produced by the method according to any one of claims 1-3.
Use of the universal monoclonal antibody according to claim 4 or 5 for the preparation of an African swine fever virus detection reagent or device.
An African swine fever virus enzyme-linked immunosorbent assay kit, characterized in that the kit comprises the universal monoclonal antibody of claim 4 or 5.
A hybridoma cell secreting a universal monoclonal antibody against an African swine fever virus strain, the accession number is CCTCC No. C2014212.
The method for preparing hybridoma cells according to claim 8, which comprises immunizing mice with purified p54 immune protein, and culturing hybrid mouse cells with mouse spleen cells to prepare hybridoma cells, and then adopting three screening methods. The antigen was used to screen positive hybridoma cell lines, and the three screening antigens were strongly positive samples were subjected to limiting dilution cell subcloning, serial hybridoma cell dilution and several consecutive monoclonal antibody tests, and the last three times. The antibody tests all the hybridoma cells with a positive rate of 100% of the pores, that is, a hybridoma cell which can stably secrete the monoclonal antibody against the African swine fever virus;

The three screening antigens include a prokaryotic expression p54 recombinant protein, a eukaryotic expression p54 recombinant protein, and a synthetic sequence of the sequence shown by Seq ID No. 1.

Seq ID No. 1: SSRKKKAAAA IEEEDIQFINPYQDQQWAEV.