质粒转染比例对人乳头瘤病毒16/18型假病毒制备的影响

Changes in Plasmid Ratios Used in Transfection Alter the Production and Infection Rate of HPV16/18 PsVs

高危型人乳头瘤病毒(Human papillomavirus,HPV)的持续感染与宫颈癌、阴道癌、口腔癌等恶性疾病的发生相关,目前主要通过基于L1蛋白的HPV疫苗进行预防。而假病毒(Pseudovirus,PsV)的高效稳定生产是相关预防性疫苗免疫保护性评价的重要环节。为初步探究PsV生产过程中各质粒的配比关系对不同型别PsV制备的影响。本实验采用人乳头瘤病毒的16型(HPV16)和18型(HPV18)两种型别的相应质粒转染细胞,编码L1蛋白的质粒转染质量固定为10μg,分别改变编码L2蛋白质粒与EGFP报告质粒的转染比例,通过计算PsV的TCID50值、测定L1蛋白含量以及PsV的感染率对不同质粒转染配比下的假病毒进行评估。结果显示在HPV16PsV模型中,L1与L2转染质量比为10μg∶2μg时,可以得到TCID50较高的PsV,而当HPV18 PsV达到最大TCID50时,L1与L2的配比为10μg∶1μg,其TCID50略大于10μg∶2μg组;L1与L2比例为10μg∶2μg时两种PsV皆可达到最大感染率;PsV模型对EGFP配比变化并不如L2敏感,L1与EGFP质量比在0.5~1之间时两种PsV均可获得较高TCID50,然而EGFP配比改变造成的感染率差异并不具有统计学意义。本研究探究了HPV16、HPV18 PsV生产的一般规律,为其他型别PsV高效生产提供指导,继而为疫苗保护性评价及结构生物学样品制备奠定基础。

The human papillomavirus(HPV)is an established carcinogen in cancer of the cervix,vagina,and oral cavity.HPV vaccines based on the L1 protein can prevent high-risk HPV infection. Highly efficient and stable production of HPV pseudoviruses(PsVs)enables evaluation of the prophylactic capabilities of HPV vaccines.To determine the optimal amount of plasmids needed for co-transfection,it is necessary to investigate the effects of changes in the plasmid ratio for PsV production. Thus,we employed the high-risk types HPV16/18 for PsV production. First,the transfection dose of the L1-plasmid was chosen to be 10μg.Then,the ratio of L2 protein or EGFP plasmids was changed,respectively. Second,median tissue culture infectious dose(TCID50),L1 concentration,and infection rate were measured to assess PsVs under different plasmid ratios. In HPV16 PsV,an optimized ratio of 10μg:2μg of L1 and L2 led to high TCID50 and the highest infection rate as well. However,in HPV 18 PsV,the optimized ratio was 10μg:1μg or 10μg:2μg to obtain the highest TCID50 and highest infection rate,respectively.Both PsVs were less sensitive to changes in the EGFP plasmid than that of L2. Each plasmid ratio of EGFP and L1 between 0.5 and 1 could produce high TCID50 HPV16/18 PsVs but subsequent changes in the infection rate were not significant.This research will contribute to highly efficient production of other types of PsVs and lay the foundation of vaccine evaluation and sample preparation for research in structural biology.