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目的构建人轮状病毒(Rotavirus,RV)vp4全基因原核穿梭表达质粒,经大肠杆菌表达验证后转化入双歧杆菌。方法从pMD19-T simple-vp4质粒中扩增RV vp4全基因,克隆至pBEX载体中,构建重组表达质粒pBEX-vp4,转化至大肠杆菌BL21(DE3)中进行表达,经SDS-PAGE和Western blot鉴定后,电转化法将重组质粒转化入双歧杆菌,提取质粒,进行PCR及双酶切鉴定。结果重组表达质粒pBEX-vp4经酶切、PCR和测序证明构建正确;在大肠杆菌BL21(DE3)中可表达相对分子质量约87 000的重组VP4蛋白,表达量较低,以包涵体形式表达,可与羊抗人轮状病毒多克隆抗体特异性结合;经PCR及双酶切鉴定,重组质粒pBEX-vp4已成功转入双歧杆菌中。结论重组表达质粒pBEX-vp4可在大肠杆菌中表达RV VP4蛋白,并可经电转化法有效地将重组表达质粒转化入双歧杆菌,为下一步在双歧杆菌中进行表达及研制基于双歧杆菌表达系统的RV重组亚单位口服活疫苗奠定了基础。
Objective To construct a prokaryotic shuttle plasmid for vp4 gene of human rotavirus (RV), which was transformed into Bifidobacterium after its expression in Escherichia coli. Methods RV vp4 gene was amplified from pMD19-T simple-vp4 plasmid and cloned into pBEX vector. The recombinant plasmid pBEX-vp4 was constructed and transformed into E.coli BL21 (DE3) for expression. SDS-PAGE and Western blot After identification, the recombinant plasmid was transformed into Bifidobacterium by electroporation and the plasmid was extracted for PCR and double enzyme digestion. Results The recombinant plasmid pBEX-vp4 was constructed correctly by restriction enzyme digestion, PCR and sequencing. Recombinant VP4 protein with a relative molecular mass of about 87 000 was expressed in E. coli BL21 (DE3) and expressed in inclusion bodies. It could specifically bind to goat anti-human rotavirus polyclonal antibody. The recombinant plasmid pBEX-vp4 was successfully transferred into Bifidobacterium by PCR and double enzyme digestion. Conclusion The recombinant plasmid pBEX-vp4 can express RV VP4 protein in E. coli. The recombinant plasmid can be transformed into Bifidobacterium by electroporation, and expressed in Bifidobacterium. Bacillus expression system RV recombinant subunit oral live vaccine laid the foundation.