EV71病毒反向遗传学系统的建立及拯救病毒的鉴定

Construction of EV71 virus reverse genetics system and identification of rescued viruses

目的:建立高效的EV71病毒反向遗传学系统,快速获得拯救病毒并鉴定其活性。方法:首先构建含有人RNA聚合酶Ⅰ启动子(PpolⅠ)、ccdB自杀基因和鼠终止子(mTer)的接收质粒pHM-ccdB,并在ccdB两侧引入AarⅠ酶切位点;为了回避EV71病毒基因组中自身的AarⅠ酶切位点,分两段PCR扩增基因组,在引物中引入必需的AarⅠ酶切位点,通过Golden Gate Clone技术连接到目的载体中获得病毒拯救质粒pHT-EV71,转染至RD细胞后,获得拯救的EV71病毒,并以RT-PCR、病毒滴度、Western blot以及电镜检测等方法鉴定拯救子代病毒。结果:通过引入ccdB致死基因和Golden Gate Clone成功构建了EV71病毒的拯救质粒(pHT-EV71),并将其转染至RD细胞后,观察到明显的致细胞病变效应(cytopathic effect,CPE),将得到的拯救子代病毒,经EV71 VP1的特异性引物进行RT-PCR扩增,观察到长约1900 bp的特异性条带;Western blot结果显示,该病毒可与EV71 VP1特异抗体结合;透射电子显微镜(transmission electron microscopy,TEM)检测可见20~30 nm球形病毒颗粒;在RD细胞中将子代病毒连续增殖8代后,检测其毒力高于母本株(6.35 lgTCID50/mL)。结论:通过引入ccdB和Golden Gate Clone技术,建立了快速、高效构建EV71病毒的拯救质粒的方法,效率达到100%,为正链RNA病毒反向遗传学系统的构建提供了一种新的策略,为进一步研究EV71病毒的致病机制及疫苗制备等提供了技术平台。

Objective:To construct an efficient reverse genetics system for EV71 virus,and to rapidly obtain the rescued viruses and identify their activity.Methods:A recipient plasmid pHM-ccdB containing human RNA polymeraseⅠpromoter,ccdB suicide gene,and murine terminator(mTer)was constructed,and the AarⅠenzyme cut site was introduced into both sides of the ccdB gene.The virus genome was amplified by PCR in two steps to avoid the AarⅠenzyme cut site contained in the EV71 genome.The necessary AarⅠenzyme cut site was introduced into the primer;then the products were connected with the target vector by Golden Gate Clone technique to obtain the rescued plasmid of EV71 virus(pHT-EV71);after the pHT-EV71 was transfected into RD cells,the rescued EV71 viruses were obtained.The progeny viruses were identified by RT-PCR,virus titer,Western blot,and electron microscopy.Results:The p HT-EV71 was successfully constructed by introducing ccdB lethal gene and Golden Gate Clone technique.After the pHT-EV71 was transfected into RD cells,obvious cytopathic effect was observed.The rescued progeny viruses obtained were amplified by RT-PCR with EV71-specific primers,and specific bands of approximately 1900 bp were observed.The viruses can be bound with EV71 VP1 specific antibody as showed by Western blot.Spherical virus particles of 20 to 30 nm in size were observed by transmission electron microscopy.After eight generations of progeny viruses in RD cells,the virulence of the progeny viruses was stronger than that of the parental viruses(6.35 lgTCID50/mL).Conclusion:A rapid and efficient method to construct the pHTEV71 with a 100%efficiency was successfully established through introducing the ccdB gene and Golden Gate Clone technique.This method provides a new strategy for the construction of reverse genetics system for positive-strand RNA viruses and a technical platform for further study on the pathogenesis and vaccine preparation of EV71 virus.