人细小病毒B19VP1基因片段原核表达载体的构建及其表达

目的　克隆人细小病毒B19　VP1基因片段,构建其重组克隆载体和表达载体,并诱导表达。方法　利用聚合酶链反应(PCR)和分子克隆技术,将B19病毒VP1基因片段扩增后,构建pGEM-T-easy克隆载体;经PCR筛选后,亚克隆于pGEX-4T-1表达载体中,并转化至BL21感受态菌;经诱导剂异丙基硫代-β-D-半乳糖苷 (IPTG)诱导表达,并以免疫印迹法对表达蛋白进行鉴定。结果　扩增出一条约 801bp的基因片段,PCR鉴定结果的与预期结果一致。经IPTG诱导,VP1融合蛋白在大肠杆菌中得到成功表达,PAGE上出现相对分子量约为 54KD的一条新生蛋白带,经蛋白印迹验证为表达蛋白B19VP1。薄层扫描现示,表达产物占菌体总蛋白的 27. 4%。结论　获得人细小病毒B19VP1基因片段的原核表达载体及其产物,对进一步研究其纯化及B19疫苗有重要意义。

Construction and expression of prokaryotic expression vector for VP1 unique region gene segment of human parvovirus B19

Objective:To clone human parvovirus B19 VP1 gene.,construct the prokaryotic expression vector,express its product Methods:VP1gene was amplified by PCR and cloned into pGEM-T-easy plasmid,.After PCR selecting.it was subcloned into pGEX-4T-1 plasmid.The recombinant expressive vector of VP1 gene was transformed into E.coli BL21,and induced to express by IPTG in BL21.Then Western blotting identified the expression product .Results:Human parvovirus B19 VP1gene was coloned.Recombinant expression plasmid pGEX-4T-1 was constructed.The expressed fussion protein was confirmed by PAGE,This product was also proved to be the target protein via Western blotting.Conclusion:The human parvovirus B19 VP1gene and its prokaryotic expression products were obtained.It might be important for study on the function of human parvovirus B19VP1 and preparing the monoclonal antibody against human parvovirus B19 VP1.