HPV18晚期蛋白L1在毕赤酵母菌中的表达及鉴定  

Expression and Identification of HPV Type 18L1 Protein in the Methylotrophic Yeast Pichia pastoris

作　　者：谢飞[1] 刘红岩 董金蓉[1] 彭建新[1] 刘勇 郭仁 杨红[1] 杨喆 

机构地区：[1]华中师范大学生命科学学院昆虫学研究所,湖北武汉430074 [2]云南沃森生物技术有限公司,云南昆明650118 [3]中国疾病预防与控制中心性病与艾滋病研究中心,北京100050

出　　处：《生物技术》2007年第3期8-11,共4页Biotechnology

基　　金：横向项目:云南沃森生物技术有限公司基金项目资助

摘　　要：目的:用毕赤酵母胞内表达载体构建含人乳头瘤病毒18型(HPV18)L1基因质粒,诱导表达并进行鉴定。方法:按照毕赤酵母密码子偏爱性原则,合成全长L1基因,然后克隆到pAO819表达载体上,在体外分别构建含一个拷贝和二个拷贝的L1基因载体。线形化后转化到GS115酵母细胞,经G418抗性筛选,获高拷贝重组子并经甲醇诱导表达,表达产物采用化学发光Western blot鉴定,一抗为抗HPV18L1蛋白鼠抗血清。结果:在55kDa处有诱导蛋白免疫印迹出现,并在电镜下观察到HPV18的病毒样颗粒(VLPs),证明该表达系统能表达出HPV18 L1蛋白。结论:本实验构建的毕赤酵母表达菌株,可经甲醇诱导表达HPV18L1晚期蛋白,为进一步研制人乳头瘤病毒18型基因工程疫苗打下基础。Objective：An expression vector of HPV type 18 L1 gene in methylotrophic yeast Pichia pastoris was constructed. HPV18- L1 protein was over- expression and identified in P. pastor/s .Methods： The whole L1 gene with preferred condons for P. pastoris was rebuilt and A - T rich regions wexe abolished.The gene was cloned into pAO819 and expression vectors with one copy and two copies of 18L1 gene were constructed in vitro. The recombinant vectors were lineared and transformed into GS115 yeast cells and screened in G418. BMGY/BMMY were used for induction and expression of L1 protein . Mouse antibody of HFV18L1 was used to identify the expressed products by western blotting. Results： Ten clones were found to produce L1 protein which can be identified with chemoluminescence Western blot. The expressed protein with a relative molecular of 55000Dr reacted with HPV antisera. The VLPs of HPV18 were observed by electron microscope.Conclusion：HPV type 18 L1 peotein was successfully expressed in P. pastaris. This study lay foundations for future animal experiments and clinical trial.

关 键 词：HPV18L1 毕赤酵母 诱导表达 

分 类 号：Q786[生物学—分子生物学] Q933