悬浮驯化HEK-293FT细胞表达人乳头瘤病毒16型假病毒及其冷冻电镜结构解析  

Preparation and Cryo-EM Structure Determination of Human Papillomavirus 16 Pseudovirion Derived from Suspension-adapted HEK293 Cells

作　　者：王大宁[1] 刘亚静[1] 郑清炳[1] 李智海[2] 范飞[2] 柳欣林 宋硕[2] 顾颖[1,2] 夏宁邵[1,2] 李少伟[1,2] 

机构地区：[1]厦门大学公共卫生学院,分子疫苗学和分子诊断学国家重点实验室,厦门361102 [2]厦门大学生命科学学院,国家传染病诊断试剂与疫苗工程技术中心,厦门361102

出　　处：《病毒学报》2016年第5期551-559,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81172885),题目:基于中和表位结构的高位HPV型交叉疫苗的分子设计

摘　　要：为了大量获得人乳头瘤病毒（HPV）16型的假病毒颗粒（PsV）,将贴壁生长的293FT细胞驯化成为悬浮细胞系进行大规模的悬浮培养,同时优化假病毒纯化方法,并使用冷冻电镜解析其三维空间结构。本研究通过逐步降低培养基中血清浓度和加入抗聚团剂等方法,获得了可在Wave生物反应器中大规模悬浮培养的293FT细胞,经过携有HPV16L1、L2基因和GFP报告基因的质粒转染,实现了大规模制备HPV16假病毒;利用CsCl等密度和蔗糖密度梯度超速离心的方法,获得高纯度的HPV16假病毒颗粒,滴度为8.2×10^5 TCID50/μL;蛋白印迹结果显示HPV16假病毒中含有L1和L2蛋白,定量ELISA测得L1蛋白的含量为156.0μg/mL;最后使用Tecnai G2F30冷冻电镜和Falcon电子直接探测相机采集冷冻HPV16假病毒的图像,应用AUTO3DEM软件解析其结构,结构分辨率为14A,呈现为T=7的等二十面体对称结构,直径为600A。本研究为HPV疫苗临床血清中和检测、高分辨率HPV16假病毒的结构解析及L1和L2相互作用研究奠定了良好基础。The goals of this study were to establish a scalable production method to prepare human papillomavirus（HPV）16pseudovirus（PsV）using suspension-adapted HEK-293 FT cells and to improve the purification efficiency of HPV PsV.Furthermore,we aimed to solve the cryo-electron microscopy（cryo-EM）structure of HPV16 PsV.The suspension f HEK-293 FT cells were generated from adherent cells by a stepwise decrease in serum content and the addition of an anti-clumping agent during culturing.The resultant HEK-293 FT suspension cells were transfected with an L1/L2 expression vector and pN31-EGFP plasmid to generate HPV16 PsV in the Wave Bioreactor.Following cell lysis,HPV16 PsV was purified by sucrose density gradient and subsequent CsCl iso-density gradient ultra-centrifugation The final titer of HPV16 PsV was 8.2 × 105 TCID50/!L.Purified HPV16 PsV was comfirmed to as contain L1 and L2protein by western blotting,and the L1 concentration was determined to be 156.0μg/mL by quantitative ELISA.Finally,a FEI Tecnai G2F30 electron microscope and AUTO3 DEM were used to solve the cryoEM structure of HPV16 PsV at a resolution of 14A.The structure shows that HPV16 PsV exists as a T=7dicosahedral lattice,with a diameter of 600A.These results will be beneficial for neutralization assays and for anti-sera for HPV vaccines,the high-resolution structure determination of HPV16 PsV,and the investigation of interactions between HPV L1 and L2.

关 键 词：人乳头瘤病毒 假病毒 悬浮培养 冷冻电镜三维结构 

分 类 号：R373.9[医药卫生—病原生物学] R373[医药卫生—基础医学]