Prokaryotic Expression and Polyclonal Antibodies Production of African Swine Fever Virus p62 Protein
This study aims to obtain the p62 protein of African swine fever virus(ASFV)for further devel-opment of serological diagnostics.According to the ASFV reference sequence in the Genbank,CP530R se-quence was synthesized and inserted into the pET-32a(+)vector,subsequently transformed into E.coli BL21(DE3)competent cell.After inducing expression by IPTG,it was identified through SDS-PAGE and Western blot.The synthesized p62 protein was purified by Ni column and immunized Balb/C female mice to The synthesized p62 protein was purified by Ni column and immunized Balb/c female mice to obtain polyclonal antibody against ASFV p62 protein.The titer of polyclonal antibody was detected by ELISA.The results suggested that the plasmid pET32a-p62 was successfully constructed.SDS-PAGE showed that the recombinant protein with 56.1 kDa was expressed as inclusion body.Western blot showed that the protein could bind to ASFV antiserum.The p62 protein was purified by Ni column,and its concentration was determined to be 2.9 mg/mL.Polycolnal antibodies were produced by immunizing mice with the puri-fied protein.The titer of antibodies reached 1∶256,000 after three rounds of immunization.The results suggested that the purified p62 protein had good antigenicity,and the polyclonal antibodies showed high reactivity and specificity,therefore laying a foundation for further study on the structure and function of p62 protein,and also on serological diagnosis of ASFV.
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