Expression,purification,analysis and application of HIV-1 core p24 antigen

Objective To express recombinant HIV-1 core p24 antigen in prokaryotic system,and investigate its antigenicity and clinical application of HIV early diagnosis.Methods HIV-1 p24 gene amplified by PCR from plasmid(PTM-1-HIV-1-gas) was subcloned into vector pET28a(+) after enzymatic digestion.The recombinant plasmid was transformed into E.coli BL21,and expressed after IPTG induction.Double enzyme digestion was used to confirm the correct insert in the recombinant plasmid.Western blotting and ELISA were used to analyze the purity and antigenicity of expressed p24 protein.The difference between recombinant p24 antigen and commercial p24 antigen were compared by time-resolved fluoroimmunoassay(TRFIA).Results PCR product and exogenous gene section from the recombinant plasmid pET28a-p24 showed the same size of 690 bp which is equal to p24 gene sequences.An exogenous expressed protein band of Mt 26×103 Mr was obtained after purified by SDS-PAGE electrophoresis.The activity and specificity of p24 protein were tested by ELISA and WB.There was no significant difference between recombinant p24 and commercial p24 antigen.Conclusion The recombinant p24 antigen constructed and expressed in E.coli had a good antigenicity same as the commercial p24 antigen.