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A kind of eukaryotic expressed African swine fever virus p72 antigen and application thereof

An African swine fever virus and expression system technology, applied in the field of eukaryotic expression of African swine fever virus p72 antigen, to achieve the effects of stable cell biological characteristics, high titer, good immunogenicity and antigenicity

Active Publication Date: 2021-12-31
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is no ASFV vaccine at present, a positive ASFV antibody indicates current or past infection, but it does not mean that there will be no ASFV vaccine in the future. Therefore, the detection of ASFV antigen is particularly important. Only by accurately determining the infection situation can we take timely measures to prevent and control the disease measure

Method used

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  • A kind of eukaryotic expressed African swine fever virus p72 antigen and application thereof
  • A kind of eukaryotic expressed African swine fever virus p72 antigen and application thereof
  • A kind of eukaryotic expressed African swine fever virus p72 antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation and identification of embodiment 1 African swine fever virus p72 recombinant protein

[0072] 1. Materials

[0073] 1.1 Strains

[0074] Escherichia coli competent DH5α and DH10Bac were purchased from Beijing Quanshijin Biotechnology Co., Ltd., pCMV vector, pFastbac1 vector, and 293F cells were preserved by the Animal Inspection and Quarantine Laboratory of the Chinese Academy of Inspection and Quarantine Science.

[0075] 1.2 Serum

[0076] The African swine fever standard positive serum inactivated by 0.3% TNBP and 1% triton X-100 was preserved by the Polish National Veterinary Institute. Porcine pseudorabies live vaccine (Kartha-K61 strain) (Pseudorabies virus, PRV) was purchased from Qingdao Yibang Bioengineering Co., Ltd.; porcine parvovirus inactivated vaccine (WH-1) (porcine parvovirus, PPV) was purchased from Wuhan Keqian Biological Co., Ltd.; porcine circovirus type 2 inactivated vaccine (LG strain) (porcine circovirus, PCV2) was purchased from H...

Embodiment 2

[0120] Embodiment 2 Preparation of anti-African swine fever virus p72 protein mouse monoclonal antibody

[0121] 1. Materials

[0122] 1.1 virus: porcine pseudorabies live vaccine (Kartha-K61 strain) (Pseudorabies virus, PRV), purchased from Qingdao Yibang Bioengineering Co., Ltd.; Porcine parvovirus inactivated vaccine (WH-1) (porcine parvovirus, PPV), Purchased from Wuhan Keqian Biotechnology Co., Ltd.; porcine circovirus type 2 inactivated vaccine (LG strain) (porcine circovirus, PCV2) was purchased from Harbin Veken Biotechnology Development Company, live swine fever vaccine (C strain) (Classical Swine Fever, CSF), highly pathogenic porcine reproductive and respiratory syndrome live vaccine (HuN4-F122 strain) (Porcine reproductive and respiratory syndrome, PRRS) were purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. The Institute of Animal Inspection and Quarantine of the Chinese Academy of Sciences isolated, identified and preserved the vaccine.

...

Embodiment 3

[0174] Example 3 Purification, Identification and Matching Test of Monoclonal Antibodies

[0175] 1. Preparation of ascites from hybridoma cell lines

[0176] Take 8-10 week-old multiparous BALB / c mice and inject 0.5 mL of special adjuvant for ascites intraperitoneally. After 12-15 days, expand the screened hybridoma cell lines, collect hybridoma cells, and centrifuge at 1000rpm for 10min. Resuspend with an appropriate amount of sterile PBS, and intraperitoneally inject 0.2mL hybridoma cells / mouse (containing about 2.5×10 61 cell), about 10-15 days later, when the abdominal circumference of the mouse increased, ascites was collected with a sterile syringe needle. Centrifuge the collected ascites at 10,000r / min for 10min, collect the middle layer, and filter it with a 0.45μm filter to obtain the corresponding monoclonal antibody (ascites). Take the supernatant respectively and press: 1:1000, 1:2000, 1:4000 , 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000 grad...

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Abstract

The invention discloses a eukaryotic expressed African swine fever virus p72 antigen and application thereof. The invention also discloses a detection kit comprising the p72 protein. The kit of the invention can specifically detect ASFV, has high sensitivity and good specificity, and provides a new detection method for clinical diagnosis and epidemiological investigation of ASFV infection.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a eukaryotic expressed African swine fever virus p72 antigen and application thereof. Background technique [0002] African swine fever (African swine fever, ASFV) is an acute, virulent, highly contagious disease of pigs caused by African swine fever virus (ASFV). swine fever (CSF) is highly similar and originated in Africa, so it is called African swine fever. [0003] In recent years, with the development of monoclonal antibody technology, the research on African swine fever monoclonal antibody has played an increasingly prominent role in ASFV research and ASFV diagnosis, mainly in two aspects. One is to combine monoclonal antibody For the establishment of immunology-related ASFV detection methods, such as direct immunofluorescence test (DIF), immunohistochemistry test, sandwich enzyme-linked immunosorbent assay, etc. for the detection of ASFV antigen; the se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C12N15/85C07K16/08G01N33/569G01N33/543
CPCC07K14/005C12N15/85C07K16/081G01N33/56983G01N33/543C12N2710/12022C12N2710/12034
Inventor 冯春燕王彩霞林祥梅吴绍强刘晓飞仇松寅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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