Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof

An African swine fever virus, monoclonal antibody technology, applied in biochemical equipment and methods, antiviral immunoglobulins, instruments, etc., can solve large regional differences, missed detection of commercial kits, missed detection of African swine fever, etc. problems, to achieve the effect of 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
[0006] The African swine fever virus genotype II strain is currently the African swine fever virus prevalent in Russia. Due to its geographical relationship, this strain poses a major threat to our country; and there is no specific detection kit for this strain at present. Commercial kits have the risk of missed detection

Method used

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  • Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof
  • Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof
  • Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 immune antigen

[0035] 1. Amplification and purification of the target gene

[0036] 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.

[0037] Seq ID No.2: 5'-CAGGGACCCGGTTATACTATTCTCATTGCTATCG-3'

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

[0039] 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

[0054] Example 2 Preparation of Screening Antigen

[0055] 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.

[0056] Among them, the artificially synthesized polypeptide shown in Seq ID No.1 was 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 is a common polypeptide sequence for all genotype II strains. Therefore, as a monoclonal antibody obtained by sc...

Embodiment 3

[0075] The preparation of embodiment 3 monoclonal antibody

[0076] 1. Animal immunization

[0077] 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.

[0078] 2. Establishment of positive hybridoma cell lines

[0079] 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 monoclonal antibody for an African swine fever virus gene II type strain as well as a preparation method and application thereof. The preparation method of the monoclonal antibody for the African swine fever virus gene II type strain 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 monoclonal antibody for the African swine fever virus gene II type strain has the advantages that the three screening antigens are adopted for screening the hybridoma cells, and especially a special artificially synthesized polypeptide for the gene II type strain is adopted as the screening antigen, so that the prepared monoclonal antibody can guarantee the detection rate of the gene II type strain, the misdetection rate is reduced, and the quarantine working quality and efficiency are improved.

Description

technical field [0001] The present application relates to the field of monoclonal antibodies, in particular to a monoclonal antibody directed against genotype II strain of African swine fever virus, and a 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, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/577G01N33/569C12N5/20
Inventor 曹琛福花群义吕建强张彩虹刘建利宗卉唐金明杨俊兴阮周曦陶虹
Owner SHENZHEN AUDAQUE DATA TECH
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