Synthesis of human enterovirus 71 VP1 and E.coli Heat-Labile Enterotoxin Subunit B Fusion gene by Overlapping PCR and its prokaryotic expression

Objective To achieve synthesis of human enterovirus 71 vp1 gene(EV71 vp1) and construction of E.coli Heat-Labile Enterotoxin Subunit B(ltb) fusion gene by Overlapping PCR,and construct its prokaryotic expression vector and test the expression product in E.coli.Method Vp1 gene was synthesized with 14 pairs of primers and the ltb gene fragment of ETEC(44815) plasmid DNA was amplified by PCR.The two fragments were linked by Overlapping PCR;the product was identified by sequencing analysis and inserted into prokaryotic expression vector pBEX.The constructed recombinant plasmid pBEX-VP1-LTB was transformed into E.coli BL21(DE3).The expression product of VP1-LTB was analyzed by SDS-PAGE and Western blotting.Then the recombinant plasmid was transformed into Bifidobacterium by electroporation.Result The vp1 gene was 891 bp and its sequence was consistent with that expected.Both PCR and restriction enzyme analysis showed that the recombinant plasmid pBEX-VP1-LTB contained the target fusion gene.The expression product with a relative molecular mass of 73 kD existed mostly in the form of inclusion body.Western bloting showed the protein had a reactionogenicity with antibody of vp1-positive sera of rabbits.The recombinant plasmids were successfully transformed into Bifidobacterium by electroporation.Conclusion The recombinant expression vector for the vp1-ltb fusion gene was successfully constructed with Overlapping PCR technique and expressed in E.coli BL21(DE3).The fusion protein contained its biological activity and this work paved the way for developing intramolecular adjuvant engineered vaccine against EV71.