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Classical swine fever antibody PPA-ELISA detection kit and preparation method thereof

A technology of swine fever antibody and swine fever virus, which is applied in the field of preparation of recombinant E2 protein of swine fever antigen, can solve the problems of difficulty in meeting the needs of swine fever diagnosis and immune detection, limited production of swine fever antigen, and low concentration of swine fever virus , easy to be labeled by horseradish peroxidase, strong antigen binding, easy to prepare

Inactive Publication Date: 2012-12-05
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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Problems solved by technology

However, at present, the antigen used in ELISA detection is cell-cultured virus, and the concentration of CSF virus in cell culture is low, resulting in limited yield and high cost of CSF antigen produced by this method, which is difficult to meet the needs of domestic CSF diagnosis and immune detection.

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  • Classical swine fever antibody PPA-ELISA detection kit and preparation method thereof
  • Classical swine fever antibody PPA-ELISA detection kit and preparation method thereof
  • Classical swine fever antibody PPA-ELISA detection kit and preparation method thereof

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Embodiment Construction

[0024] 1. Extraction of viral genome RNA Add 1 ml of TRIzol reagent to 200 μl of CSFV virus liquid, and extract viral genome RNA according to the instructions of TRIzol reagent. TRIzol was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0025] 2. Synthesis of cDNA by reverse transcription Using the extracted viral genome RNA as a template, first reverse transcribe into cDNA. The reverse transcription reaction was operated according to the 20μl system. Take 10μl extracted RNA, 2.5μl downstream primer (5'-CGGAATTCGAAGATTACAGGTATGCGATA-3'), 4μl 5×M-MLV Buffer, 0.5μl Ribonuclease Inhibitor, 2μl 2.5mM dNTP, 1μl M-MLV For reverse transcriptase, after 50 minutes at 42°C, inactivate at 95°C for 5 minutes. M-MLV reverse transcriptase, Ribonuclease Inhibitor, and dNTP were purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0026] 3. PCR amplification Use cDNA as a template for PCR reaction. The PCR reaction system is: 0.5μl Ex Taq (5U / μl), 5μl ...

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Abstract

The present invention relates to a classical swine fever antibody PPA-ELISA detection kit and a preparation method thereof. The kit preparation comprises: adopting RT-PCR to amplify a 550 bp gene fragment of E2 gene, wherein the fragment contains 4 neutralizing antigen regions of A, B, C and D; cloning the amplified 550 bp gene fragment into a pMD18-T vector; carrying out identification, wherein the identification result is correct; further directionally cloning the fragment into a PGEX-6P-1 prokaryotic expression vector; transforming BL21 expression bacterial; carrying out IPTG induced expression to obtain recombinant protein expressed in a form of inclusion body; adopting an affinity chromatography method to separate and purify the recombinant protein; adopting immunoblotting to detect antigenicity and specificity of the purified protein; and adopting the recombinant protein as coating antigen, adopting horseradish peroxidase-labeled staphylococcal protein A (SPA) as second antibody, optimizing reaction conditions of indirect ELISA, establishing an indirect ELISA detection method for detection of classical swine fever virus antibody, and assembling the classical swine fever antibody PPA-ELISA detection kit.

Description

technical field [0001] The invention relates to a method for preparing recombinant E2 protein of swine fever antigen and a method for preparing a PPA-ELISA detection kit for swine fever antibody by using the recombinant protein. Background technique [0002] Classical swine fever (CSF) is a highly contagious and fatal infectious disease of pigs caused by classical swine fever virus (CSFV). Clinically, it is characterized by persistent high fever and massive bleeding on the skin and mucous membranes. Points are the main features. Swine fever is highly contagious, mainly transmitted through the respiratory tract and digestive tract, regardless of season, spreads quickly, has high morbidity and mortality, and is widespread. It has caused huge economic losses to the pig industry and is a serious threat to my country's pig farming. one of the most important diseases of the industry. The World Organization for Animal Health lists it as a class A infectious disease, and my country...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/40C12N15/70C07K14/08G01N33/569G01N33/543
Inventor 沈志强王金良李娇祖立闯
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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