摘要
目的 构建人乳头瘤病毒(human papillomavirus, HPV)16型(HPV16)和18型(HPV18)假病毒,探索优化假病毒制备的条件,并利用制备的假病毒评价HPV疫苗血清抗体的中和活性。方法 将优化后的HPV16和HPV18 L1、L2和EGFP基因片段分别插入pcDNA3.1+载体中,通过多质粒共转染293FT细胞制备假病毒。对转染试剂、多质粒共转染比例、转染时间和细胞裂解时间进行摸索优化,通过测定假病毒的滴度,确定假病毒制备的最佳条件。收获的假病毒用30%的碘克沙醇作为介质进行超速离心纯化,纯化后的假病毒用于电镜观察。利用假病毒中和试验对制备的原核表达复性蛋白HPV16 L1免疫血清以及HPV16和HPV18病毒样颗粒(virus-like particles, VLP)免疫血清进行中和活性评价。结果 成功构建了假病毒,并确定了假病毒制备的最佳条件:pL1∶pL2∶pEGFP等3种质粒共转染比例为1∶0.1∶0.5,总量为4μg;转染时间为72 h,细胞裂解时间为24 h时获得的假病毒滴度最高。透射电镜观察结果显示,HPV16和HPV18假病毒颗粒基本呈规则圆形,形态上与天然病毒颗粒相似。中和试验结果显示,当用原核表达复性蛋白HPV16 L1免疫血清中和HPV16假病毒时,并不能抑制假病毒的感染活性;但HPV16和18 VLP免疫血清能明显的抑制HPV16和HPV18假病毒的感染活性。结论 成功构建并制备了2种高危型HPV假病毒,并可用于中和试验评价HPV疫苗血清抗体的中和活性。
Objective The aim of this study is to construct pseudoviruses for human papillomavirus (HPV) types16 and HPV18, to search for the optimal conditions of preparating pseudovirions which was used in detection of the serum neutralizing antibodies produced from HPV vaccine immunization.Methods The optimized HPV16 and HPV18 L1, L2 and EGFP genes were inserted into pcDNA3.1+ vectors, respectively.The co-transfection of three placmids into 293FT cells was used to make pseudoviruses.The optimal transfection reagent, the co-transfection ratio of multiple plasmids, and the time of cell harvesting and cell lysis were investigated, on which the optimal conditions in preparation of pseudoviruses were determined by detecting the titer of pseudovirus.The raw pseudoviruses were purified by ultra-centrifugation using30% iodixanol as a medium and then visualised with an electron microscope.The evaluation was carried out in serum antibodies against HPV16 L1 renaturation protein expressed in prokaryotic cells and HPV16 and HPV18 virus-like particles (VLP) were measured by pseudovirus neutralization assay.Results The pseudoviruses were constructed and the optimal conditions for pseudovirus preparation were determined as followings: the co-transfection ratio of pL1∶pL2∶pEGFP (enhanced green fluorescent protein) was1.0∶0.1∶0.5 and the total amount was4 μg;the cells were harvested at 72 hours;the cell lysis time was 24 hours.Under these conditions, the highest titer was obtained from prepared pseudovirus.The transmission electron microscopy showed that the pseudoviral particles of HPV16 and HPV18 were basically in a round shape and similar to those of natural viruses.The neutralization assay indicated that the infection activity of pseudovirus could not be inhibited by serum antibody against HPV16 L1 renaturation protein expressed in prokaryotic cells, but can be inhibited by the serum against HPV16 and HPV18 VLP.Conclusion Two high-risk HPV pseudoviruses were successfully constructed and prepared, which can be used to measure the serum neutralization antibodies produced from HPV vaccine immunization.
作者
李雄雄
董占柱
吴元元
安静
马超
LI Xiong-xiong;DONG Zhan-zhu;WU Yuan-yuan;AN Jing;MA Chao(Second Laboratory of Research, Lanzhou Institute of Biological Products Co., Ltd., Center forGansu Provincial Vaccine Engineering Research, Lanzhou 730046, Gansu Province, China)
出处
《微生物学免疫学进展》
2019年第5期1-9,共9页
Progress In Microbiology and Immunology
关键词
高危型人乳头瘤病毒
假病毒
中和试验
感染活性
High-risk human papillomavirus
Pseudovirus
Neutralization assay
Infection activity
