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African swine fever virus blocking ELISA antibody detection kit and its preparation method and application

A technology of African swine fever virus and kit, which is applied in the field of African swine fever virus blocking ELISA antibody detection kit and its preparation, can solve the problem of unsuitable for large-scale rapid detection, high cost of African swine fever antigen, and African pigs without vaccine plague and other problems, to achieve the effect of fast detection, low cost and high sensitivity

Active Publication Date: 2022-03-11
杭州恒奥科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no vaccine on the market for the prevention and control of African swine fever. The cost of detecting African swine fever antigen is high and the speed is slow, which is not suitable for rapid detection in large quantities.

Method used

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  • African swine fever virus blocking ELISA antibody detection kit and its preparation method and application
  • African swine fever virus blocking ELISA antibody detection kit and its preparation method and application
  • African swine fever virus blocking ELISA antibody detection kit and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 African swine fever virus P30 protein

[0022] The codon-optimized nucleotide sequence of the African swine fever virus P30 protein (the specific sequence is shown in SEQ ID NO.1) is cloned into a eukaryotic expression vector (such as ). The nucleotide sequence of the codon-optimized African swine fever virus P30 protein (the specific sequence is shown in SEQ ID NO.1) was used as a template, and EcoRI and HindⅢ double restriction sites were used to connect to construct pEE12.4-P30 -6His expression plasmid. The recombinant plasmids identified as positive were sent to Huada Biological Company for sequence determination, and software was used to analyze and compare the determined nucleotide sequence and encoded amino acid sequence to check the correctness of the reading frame.

[0023] The correctly identified pEE12.4-P30-6His expression plasmid was transfected into CHO-K1 cells. Pressurized screening started 24 hours after transfection: T...

Embodiment 2

[0025] Embodiment 2 Preparation of Anti-African Swine Fever Virus P30 Protein Monoclonal Antibody

[0026] The African swine fever virus P30 recombinant protein prepared in Example 1 was used to immunize 8-week-old BALB / c mice. During the first immunization, the African swine fever virus P30 recombinant protein was emulsified with an equal volume of Freund's complete adjuvant, and the mice were inoculated intraperitoneally. 100 μg protein / mouse; 7 days later, African swine fever virus P30 recombinant protein was emulsified with an equal volume of Freund’s incomplete adjuvant, and the mice were immunized by the second intraperitoneal inoculation route, 100 μg protein / mouse; 7 days later, the third mouse peritoneal route was directly Immune African swine fever virus P30 recombinant protein, 100 μg / monkey; on the third day after immunization, take mouse splenocytes and mouse myeloma cell SP2 / 0 fusion, and culture in HAT selective medium; 10 days later, African swine fever virus The...

Embodiment 3

[0030] Example 3 Preparation of African swine fever virus blocking ELISA antibody detection kit

[0031] The African swine fever virus blocking ELISA antibody detection kit developed in this research, on the basis of successful trial production in the laboratory, has continuously produced 5 batches of kits in the GMP workshop of the company's diagnostic reagents, and the batch numbers are 210305, 210310, and 210315. , 210320, 210325, a total of 100 boxes were produced, and the quantity of each batch of products was 20 boxes, and the specification was 2 boards / box; Strict inspection was carried out on the samples of 5 batches of kits produced, and the results all met the requirements, summarized as follows:

[0032] 1 material

[0033] 1.1 The batch number of ASFV P30 protein used for coating is: 201102, the protein content is 1.215mg / ml, which is produced, identified, kept and supplied by our company.

[0034] 1.2 The batch number of the enzyme-labeled antibody is: 201002, an...

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Abstract

One aspect of the present invention provides an African swine fever virus blocking ELISA antibody detection kit, said kit is coated with African swine fever virus P30 protein on a 96-well microtiter plate, and said African swine fever virus P30 protein consists of Expressed by CHO cell line, the relative molecular weight of the African swine fever virus P30 protein is 36kD, and the coding sequence of the African swine fever virus P30 protein is shown in SEQ ID No.1. On the other hand, the present invention also provides the application of the African swine fever P30 protein and the anti-ASF virus P30 protein monoclonal antibody in the preparation of diagnostic reagents. The African swine fever virus blocking ELISA antibody detection kit provided by the present invention is suitable for the detection of pig serum, has strong specificity, high sensitivity, good stability, and fast detection speed, and can be used for early screening of African swine fever virus. It is especially suitable for on-site African swine fever infection diagnosis, epidemiological investigation and quarantine inspection of pig international trade.

Description

technical field [0001] The invention belongs to the field of virus epidemic disease diagnosis technology and animal quarantine, and in particular relates to an African swine fever virus blocking ELISA antibody detection kit and a preparation method and application thereof. Background technique [0002] African swine fever (English name: African Swine fever, referred to as ASF) is caused by African swine fever virus (English name: African Swine fever virus, referred to as ASFV) that infects domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.) Cause an acute, hemorrhagic, severe infectious disease. The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08
CPCG01N33/577G01N33/56983G01N33/581C07K14/005C07K16/081C12N15/85G01N2469/20G01N2333/01C07K2317/56C12N2710/12022C12N2800/22C12N2800/107
Inventor 查银河孙统威徐慧珍周晶晶张稳涛余晓玉
Owner 杭州恒奥科技有限公司
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