用甲醇酵母表达经基因优化的HPV 6型L1蛋白

Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris

目的利用甲醇酵母系统Pichia pastoris高效表达HPV 6型L1蛋白。方法按照Pichiapastoris偏爱密码子合成L1全长基因,构建pPIC3.5K-HPV6-L1表达载体,转化KM71,经组氨酸缺陷的MD培养基和G418筛选,PCR确认L1基因整合,使用BMGY/BMMY培养/诱导目的基因的表达。结果筛选到3株阳性表达克隆,Western blot显示表达产物有部分糖基化现象,使用能识别完整VLP的单抗进行间接免疫荧光检测提示L1蛋白在Pichia细胞内以空间构象形式存在。通过离子交换和亲和层析两步纯化从1L发酵液中得到125μg纯的L1蛋白。结论通过基因优化在甲醇酵母中表达HPV6-L1蛋白,这将为结构与功能研究以及疫苗开发提供条件。

Objective 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. Methods The whole L1 gene with preferred codons for P.pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5 K, electroporation of KM71, in vivo screen of multiple inserts by G418 resistanc, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins. Results Three clones were found to produce L1 protein which can be identified with Western blot.Compared with L1 protein from E. coll, 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 125μg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography. Conclusion HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.