【摘要】 目的:研制针对我国宫颈癌高危相关的HPV16型的治疗性无佐剂蛋白疫苗。方法:应用PCR技术自我国山西宫颈癌高发现场分离到的毒株——HPV16z中获得E6/E7转化基因片段,自卡介苗菌株中克隆获得Hsp65基因片段,对E6/E7基因片段定点突变修饰,构建pET28a-Hsp65-E6/E7表达载体,在大肠杆菌BL21(DE3)中表达融合蛋白,并研究重组蛋白的纯化方案和工艺。结果:成功构建pET28a-Hsp65-E6/E7重组表达载体,E6/E7突变位点正确,融合蛋白在亲和层析柱上正确复性和初步纯化,经阴离子交换色谱纯化后蛋白纯度达到95%。结论:该研究为无佐剂治疗性重组蛋白疫苗Hsp65-E6/E7的进一步功能研究奠定了基础。更多还原

【Abstract】 Objective: To develop a therapeutic adjuvantfree protein vaccine against HPV16 which is closely related to cervical cancer of China. Method: First the E6/E7 gene by PCR technology from HPV16z virus strain was isolated in the highrisk cervical cancer area of Shanxi province of China in1990s, and again got the gene segment of Hsp65 from BCG by the same method, mutated the transforming codes in sequences of HPV 16 E6/E7 genes and thus constructed the expression vector pET28aHsp65E6/E7, expressed the Hsp65E6/E7 fusion protein in E.coli BL21(DE3) strain and researched optimal protein purification procedures. Results: The expression vector pET28aHsp65E6/E7 was constructed successfully and E6/E7 gene was mutated correctly. Hsp65E6/E7 fusion protein was renatured and purified on the affinity chromatography column simultaneously. The protein purity achieved 95% after the anionic exchange chromatograph purification. conclusions: This research laid a foundation for further functional study of the therapeutic adjuvantfree protein vaccine——Hsp65E6/E7.更多还原