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Method for preparing soluble E2 antigen for detecting swine fever virus serum antibody

A serum antibody, swine fever virus technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, viral peptides, etc., can solve the problems of restricting the application of recombinant proteins, and achieve the goal of overcoming false positives, promoting expression, and achieving good results. Effect

Active Publication Date: 2011-08-31
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This severely limits the application of recombinant proteins in scientific research

Method used

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  • Method for preparing soluble E2 antigen for detecting swine fever virus serum antibody
  • Method for preparing soluble E2 antigen for detecting swine fever virus serum antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] 1. Cloning of E2 gene antigen domain part

[0082] 1) Design expression primers

[0083] Design a pair of specific oligonucleotide expression primers uE and dE, and add restriction endonuclease sites at the 5 ends respectively, the sequence is:

[0084] uE: 5'-GC GAATTC GGACTCACCACTACATGC-3'EcoRI

[0085] dE: 5'-GC CTCGAG TAGCCATCTGTGCTGATTC-3’Xhol I

[0086] 2) Total RNA extraction: use tissue lysate (TRIZOL reagent) to extract total RNA from the spleen tissue of rabbits infected with the CSF rabinized virus vaccine strain.

[0087] 3) Synthesis of cDNA by reverse transcription: Take 5~10 μl of the extracted RNA and add it to a 20 μl reverse transcription system containing 1×RT buffer (50mM Tris-HCl (pH8.3), 75nM KCl, 8mMMgCl 2 , 10mMDTT), dNTP (each 10mM), primer dE50pmol, AMV RTase 10U, RNase inhibitor 20U. Water bath at 42°C for 1 hour, then boil for 5 minutes, and store at -20°C for later use.

[0088] 4) PCR amplification of the E2 gene: Take 5 μl of the ...

Embodiment 2

[0125] The method of Example 1 was repeated, except that in step 5, the induction temperature for large-scale induction of expression was 20° C., and the induction time was 12 hours.

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Abstract

The invention relates to a method for preparing a soluble E2 antigen for detecting a swine fever virus serum antibody. The method comprises the following steps: A. a structural domain part of the E2 gene antigen is cloned; B. a recombining expression vector is constructed; C. the recombining expression vector is expressed in a LB cultivating substrate to obtain soluble recombining E2 protein; D. the recombining E2 protein is subjected to affinity chromatography and purification; E. pGST-E2 expression protein is identified; and the recombining E2 protein is identified and expressed through SDS-PAGE and Western Blotting; and F. recombining E2 protein is utilized to build an indirect ELISA method for detecting the swine fever virus serum antibody. The method improves the prior LB cultivatingsubstrate, promotes the expression of the soluble E2 protein and solves the difficult problems that the swine fever virus E2 antigen in colibacillus is mainly expressed by an inclusionbody; the obtained soluble E2 protein has similar immunological effect with a full swine fever virus particle and can be used as the antigen to substitute for a swine fever virus particle, thereby establishing the sensitive, special, safe and reliable indirect ELISA method for detecting the swine fever virus serum antibody.

Description

technical field [0001] The invention relates to a method for preparing E2 protein as an antigen, in particular to a method for preparing a soluble E2 antigen protein for detecting serum antibodies to swine fever virus. Background technique [0002] Classical swine fever (CSF) is caused by classical swine fever virus (CSFV), which seriously threatens the pig industry and is one of the economically significant severe viral diseases. First, it is also listed as a first-class infectious disease in my country, which can lead to the death of pigs at various growth stages and cause huge economic losses to the pig industry. CSFV is an enveloped RNA virus with a genome length of about 12.3kb, containing a large open reading frame (ORF), which is translated into a poly precursor protein containing about 3898 amino acid residues, of which C, Erns, E1, E2 is a structural protein. As one of the main structural glycoproteins on the virion envelope, CSFV E2 protein is the main inducer of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/70C07K14/08G01N33/53
Inventor 刘湘涛尹双辉孙世琪田宏尚佑军韩雪清刘艳红
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI