Li Ping-chuan, Zhang Xiao-guang, Zhou Ling, Zeng Yi
Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2003 Dec;17(4):310-4.
Human papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.
The whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.
Three clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.
HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.
人乳头瘤病毒6型（HPV 6）可引发尖锐湿疣，这是一种常见的性传播疾病。L1衣壳蛋白是一种极具潜力的疫苗候选物，已进入二期临床试验阶段。但现有的在昆虫细胞中生产L1衣壳的方法对于发展中国家而言成本过高。作为甲基营养型酵母，毕赤酵母表达系统具有经济性且表达水平高。本研究旨在在毕赤酵母中过表达HPV6-L1蛋白。
重建了带有毕赤酵母偏好密码子的完整L1基因，并去除了富含A-T的区域，将其克隆到pPIC3.5K中，电穿孔导入KM71，通过G418抗性在体内筛选多个插入片段，并对毕赤酵母整合体进行PCR分析，使用BMGY/BMMY诱导和表达L1蛋白。
发现三个克隆可产生L1蛋白，通过蛋白质印迹法可对其进行鉴定。与大肠杆菌产生的L1蛋白相比，毕赤酵母产生的L1具有一些糖基化修饰。在间接免疫荧光试验中与单克隆抗体H6B10.5强烈反应表明，毕赤酵母细胞中表达的L1蛋白保留了其天然构象表位，这对于疫苗应用很重要。通过离子交换和镍-氮三乙酸亲和层析，从1升培养物中总共可获得125微克纯L1蛋白。
在毕赤酵母中表达的HPV 6型L1蛋白将有助于HPV疫苗的研发及结构-功能研究。
