Prokaryotic expression of equine herpesvirus type 1 ORF2 protein and preparation of its polyclonal antibodies
The purpose of this study was to clone the ORF2 gene of equine herpesviruses type 1(EHV-1),obtain the ORF2 protein by Escherichia coli prokaryotic expression system,and prepare polyclonal antibodies.According to the whole genome sequence of EHV-1 YM2019 strain(GenBank:MT063054),a pair of primers were designed,and ORF2 gene was cloned into pET-28a-ORF2 prokaryotic expression vector.ORF2 protein was induced by Transetta(DE3)competent cells and IPTG,purified by Ni-NTA agarose purification resin,and immunized with balb/c mice to detect the titer and specificity of polyclonal antibody.The results showed that the ORF2 gene was successfully cloned into the prokaryotic expression vector pET-28a,and the ORF2 protein was obtained by Escherichia coli expression system.The size of ORF2 protein was about 38 ku,and it existed in the form of inclusion body.The recombinant protein with high purity obtained by Ni-NTA resin can specifically bind to EHV-1 positive serum.The titer of polyclonal antibody detected by indirect ELISA was 1:64 000,and ORF2 protein expressed by RK-13 could be specifically recognized by polyclonal antibody.The study indicated that the recombinant ORF2 protein expressed by Escherichia coli had good reactivity,and the polyclonal antibody prepared by ORF2 protein had high titer and specificity,which could lay a foundation for the study of biological function of ORF2 protein and gene deletion vaccine.
Equine herpesvirus type 1ORF2 proteinProkaryotic expressionPreparation of polyclonal antibody
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