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General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof

An African swine fever virus and monoclonal antibody technology, applied in the field of general monoclonal antibodies, can solve the problems of African swine fever missed detection and large regional differences, and achieve the effect of reducing missed detection and improving work quality and efficiency

Inactive Publication Date: 2015-04-08
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large regional differences among African swine fever virus strains, the existing commercial kits are prone to missed detection of African swine fever caused by certain strains

Method used

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  • General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof
  • General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof
  • General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 immune antigen

[0036] 1. Amplification and purification of the target gene

[0037] Design a pair of primers 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 is provided by the Shenzhen Entry-Exit Inspection and Quarantine Bureau Animal and Plant Inspection and Quarantine Technology Center, The plasmid contains a 552bp p54 gene of the sequence shown in Seq ID No.10. The upstream primer of the primer is the sequence shown in Seq ID No.2, and the downstream primer is the sequence shown in Seq ID No.3.

[0038] Seq ID No.2: 5'-CAGGGACCCGGTTATACTATTCTCATTGCTATCG-3'

[0039] Seq ID No.3: 5'-GGCACCAGAGCGTTCAAGGAGTTTTTCTAGGTC-3'

[0040] After the above primers were synthesized by the primer synthesis company, PCR amplification was performed 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, an...

Embodiment 2

[0055] Example 2 Preparation of Screening Antigen

[0056] In this example, three screening antigens are used to screen hybridoma cells. One is the prokaryotic expression of p54 recombinant protein as the immune antigen in Example 1, and the p54 immune protein purified by nickel column is used as the screening antigen; the other is artificial synthesis The polypeptide shown in Seq ID No.1 synthesized by the method; the third is to use the pFastBac / NT-TOPO insect expression system to eukaryotically express the p54 recombinant protein, and use the sonicated supernatant of the infected recombinant baculovirus cells as the screening antigen.

[0057] Among them, the artificially synthesized polypeptide shown in Seq ID No.1 is proposed in this case on the basis of comparing the p54 protein sequences of all African swine fever viruses that have been published so far, and it is a common polypeptide sequence of all African swine fever viruses. Therefore, As a screening antigen, it can...

Embodiment 3

[0076] The preparation of embodiment 3 monoclonal antibody

[0077] 1. Animal immunization

[0078] The purified p54 immune protein prepared in Example 1 was mixed and emulsified with an equal amount of Freund's complete adjuvant, and subcutaneously injected into 7-week-old BALB / c female mice, 0.2 μg each. After 14 days, the p54 protein was mixed and emulsified with an equal amount of Freund's incomplete adjuvant, and injected subcutaneously at multiple points. After 21 days, the p54 protein was mixed and emulsified with an equal amount of Freund's incomplete adjuvant, and injected subcutaneously at multiple points. A booster injection without any adjuvant was carried out 3 days before the planned and SP2 / 0 cell fusion.

[0079] 2. Establishment of positive hybridoma cell lines

[0080] Take the immunized mice with high titer, mix their splenocytes with SP2 / 0 myeloma cells at a ratio of 10:1, and prepare hybridoma cells by conventional PEG fusion method. The screening anti...

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Abstract

The invention discloses a general monoclonal antibody for African swine fever virus strains as well as a preparation method and application thereof. The preparation method of the general monoclonal antibody for the African swine fever virus strains comprises the following steps: immunizing a mouse by adopting 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 which can stably secrete a monoclonal antibody, and then preparing the monoclonal antibody by adopting 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. The preparation method of the general monoclonal antibody for the African swine fever virus strains has the advantages that the three screening antigens are adopted for screening the hybridoma cells, and especially an artificially synthesized common polypeptide of all the African swine fever virus strains is taken as the screening antigen, so that the prepared general monoclonal antibody can be used for detecting strains in different geographic regions, the misdetection rate caused by regional difference among the strains is reduced, and the quarantine working quality and efficiency are improved.

Description

technical field [0001] The application relates to the field of monoclonal antibodies, in particular to a general monoclonal antibody for African swine fever virus strains, and the preparation method and application of the monoclonal antibody. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV), and its clinical symptoms are high fever, skin congestion, Abortion, edema and organ hemorrhage, the fatality rate is as high as 100%. However, after a small number of strains infected domestic pigs, subacute infection or recessive infection occurred in pigs. The World Organization 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 our country. Our country has stipulated this disease as a first-class animal infectious disease, and it has been studied as ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/70G01N33/577G01N33/569C12N5/20C12R1/91
CPCC07K16/08C12N1/00C12N15/70C12N15/85C12R2001/91
Inventor 曹琛福花群义刘建利唐金明吕建强宗卉张彩虹杨俊兴孙洁廖立珊
Owner SHENZHEN AUDAQUE DATA TECH
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