Prokaryotic Expression of the VP3 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies
In order to prepare polyclonal antibodies against VP3 protein of Senecavirus A(SVA),a recombinant expression plasmid named as pET-SVA-VP3 was constructed through cloning SVA VP3 gene into a prokaryotic expression vector known as pET-28a using homologous recombination technology,and transformed into Escherichia coli competent cell BL21(DE3)to express VP3 protein by IPTG induction at a concentration of 1 mmol/L,and the expressed protein was purified,then the purified VP3 recombinant protein was injected into New Zealand white rabbits via subcutaneous multi-point to prepare polyclonal antibodies,followed by evaluation on its titer,reactivity and specificity by indirect ELISA,indirect immunofluorescence assay(IFA)and Western blot.The results showed that the VP3 recombinant protein was expressed in a form of inclusion body with a molecular mass of about 36 kDa;the prepared antibodies could specifically react with VP3 recombinant protein and SVA,and the titer evaluated by ELISA reached up to above 1:10 000,and those by Western blot and IFA were both 1:5 000.In conclusion,SVA VP3 polyclonal antibodies were successfully prepared,which were with high titer and specificity.The study laid a foundation for future researches on the pathogenic mechanism and detection of SVA.
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