Prokaryotice expression of the NS1 gene of PPV and renaturationof the recombinant protein

Abstract:

The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1.The insert position，the size and the frame were identified by PCR，restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37℃ induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body.Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.