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目的 构建一种诱导肿瘤细胞凋亡的蛋白质的原核表达系统 ,以制备抗原物质凋亡素融合蛋白。方法 通过PCR的方法 ,以pcDNA -VP3质粒为模板 ,扩增出凋亡素VP3基因。将其克隆到原核表达载体 pET DsbA的多克隆位点 ,构建成凋亡素的高效原核表达载体 pET DsbA VP3,将该质粒转化到大肠杆菌E .coliBL2 1 (DE3) plysS中 ,以异丙基硫代 β D 半乳糖苷 (isopropylthio β D galactoside,IPTG)对其进行诱导表达 ,聚丙烯酰胺凝胶电泳分析目的蛋白。 结果 转化有凋亡素原核表达载体pET DsbA VP3的大肠杆菌E .coliBL2 1 (DE3) plysS经IPTG诱导后 ,聚丙烯酰胺凝胶电泳出现 38 3KD的目的蛋白条带。结论凋亡素原核表达载体 pET DsbA VP3能高效表达出凋亡素融合蛋白
OBJECTIVE: To construct a prokaryotic expression system of a protein that induces apoptosis in tumor cells to prepare the apoptin fusion protein. Methods The apoptin VP3 gene was amplified by PCR and pcDNA-VP3 plasmid was used as a template. Cloned into the multi-cloning site of the prokaryotic expression vector pET DsbA to construct the prokaryotic expression vector pET DsbA VP3 of apoptin. The plasmid was transformed into E.coli BL21 (DE3) plysS, The recombinant protein was induced by isopropylthio β D galactoside (IPTG) and analyzed by polyacrylamide gel electrophoresis. Results After induced by IPTG, the E. coli E. Coli BL21 (DE3) plysS transformed with apoptin prokaryotic expression vector pET DsbA VP3 showed a 383KD target protein band on polyacrylamide gel electrophoresis. Conclusion Prokaryotic expression vector pET DsbA VP3 can express apoptin fusion protein