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/!L;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14,呈现为T=7的等二十面体对称结构,直径为600。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 14.The structure shows that HPV16 PsV exists as a T=7dicosahedral lattice,with a diameter of 600.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.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
