重叠延伸PCR法合成EV71 VP1-LTB融合基因及其原核表达

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

目的用重叠延伸PCR(overlap extension PCR)法获得人肠道病毒71型vp1与大肠埃希菌不耐热肠毒素B亚单位(ltb)的融合基因,构建EV71 VP1-LTB融合表达质粒并在原核系统表达。方法设计14对引物通过重叠延伸PCR技术合成vp1基因,以ETEC(44815)质粒DNA为模板,PCR扩增ltb基因,将vp1基因与ltb基因连接并测序,连接产物插入原核表达质粒pBEX,构建重组质粒pBEX-VP1-LTB,转化E.coliB1221(DE3)进行表达,SDS-PAGE及W estern blotting分析其表达。电转法将重组质粒转入双歧杆菌。结果合成的vp1基因全长891 bp,测序结果与预期相符,重组表达质粒pBEX-VP1-LTB经PCR及双酶切鉴定,表明构建正确;目的蛋白在E.coliBL21(DE3)中获得了表达,Western bloting分析结果表明该蛋白具有与EV71 VP1抗体的反应原性;电转法成功将重组质粒转入双歧杆菌。结论应用重叠延伸PCR技术成功构建了EV71 VP1-LTB融合表达质粒,并在E.coliBL21(DE3)中获得有生物学功能的VP1表达产物,重组质粒双歧杆菌转化及VP1-LTB融合蛋白的表达研究,为研制EV71分子内佐剂疫苗打下了基础。

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.