猪硒蛋白W的原核表达及IgY多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2019.06.028

Abstract

Abstract:

The aim of the experiment was to optimize the prokaryotic expression system of selenoprotein W (SelW),and evaluate the immunogenicity of SelW and the feasibility of detecting SelW in swine based on IgY antibodies.The obtained gene sequence codon of porcine SelW was optimized,synthesized and ligated into pET-32a(+) expression vector,and the prokaryotic expression recombinant plasmid pET32a(+)-SelW was constructed and transformed into E.coli BL21 host strain,which was induced by IPTG.The expression of SelW in recombinant swine was identified by SDS-PAGE,and the IgY polyclonal antibody against SelW was prepared by immunizing laying hens,the IgY antibody titer was detected by indirect ELISA,the specificity of the IgY recognition antigen was examined using Western blotting.The results of SDS-PAGE showed that SelW gene was successfully expressed in prokaryotic cells,and a recombinant protein with a size of about 33 ku was obtained.The optimal expression concentration and time of IPTG were 0.5 mmol/L and 6 h,respectively.Soluble analysis results showed that the recombinant protein existed mainly in the form of inclusion bodies.The results of indirect ELISA showed that the titer of IgY polyclonal antibody at 45 days after immunization could reach 1:51 200.Western blotting results showed that the recombinant protein had good immunogenicity,and the prepared IgY antibody had higher affinity with SelW.This experiment successfully constructed and optimized the prokaryotic expression system of SelW,increased the expression of SelW,and obtained SelW with good immunogenicity,and the prepared IgY antibody specifically recognized SelW in swine muscle tissue,which could be used to detect the expression of SelW in swine tissues.By measuring the expression of SelW to reflect the level of selenium in swine,it could be used to prevent selenium poisoning and selenium-deficient disease monitoring,and laid a foundation for further exploration of SelW biological function.

Key words: swine; selenprotein W(SelW); prokaryotic expression; IgY polyclonal antibody

CLC Number:

S828

ZHOU Jiping, CAO Ruimin, MIKEREMU Shayibuzhati, JIANG Zhihui, ZHANG Xiaoying. Prokaryotic Expression of Porcine Selenoprotein W and Preparation of IgY Polyclonal Antibody[J]. , 2019, 46(6): 1801-1807.

References

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Prokaryotic Expression of Porcine Selenoprotein W and Preparation of IgY Polyclonal Antibody

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