细胞嗜性差异的2株柯萨奇病毒A10型的构建与拯救  

Construction and rescue of two Coxsackievirus A10 strains with different cell tropism

作　　者：裴捷 杨祥 汪梦俊 刘睿伦 周金戈 吕诗韵 王文辉 郭靖 孟胜利 申硕 PEI Jie;YANG Xiang;WANG Meng-jun;LIU Rui-lun;ZHOU Jin-ge;LV Shi-yun;WANGWen-hui;GUO Jing;MENG Sheng-li;SHEN Shuo(Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products,National Engineering Technology Research Center of Combined Vaccines,Vaccine Technology Innovation Center of Hubei Province,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究一室国家联合疫苗工程技术研究中心湖北省疫苗技术创新中心,湖北武汉430207

出　　处：《中国生物制品学杂志》2022年第12期1437-1442,共6页Chinese Journal of Biologicals

基　　金：国家“重大新药创制”科技重大专项(2015ZX09102021);湖北省技术创新重大专项(2017ACA078)。

摘　　要：目的以Vero细胞及人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞嗜性差异的柯萨奇病毒A10型(Coxsackievirus A10,CV-A10)2个毒株为模板,分别构建感染性克隆,并完成病毒拯救,为CV-A10毒株的细胞嗜性研究奠定基础。方法分别扩增Vero^(+)/RD+及Vero^(-)/RD+细胞嗜性差异的CV-A10毒株基因组,在5′端引入T7启动子序列,3′端引入polyA尾及NotⅠ酶切位点。将扩增得到的基因组序列与线性化pBR322载体以无缝连接技术连接得到全长质粒,经线性化后分别与T7-RNA聚合酶表达质粒共转染RD细胞完成病毒拯救。通过病毒感染后细胞形态观察及间接免疫荧光试验,比较拯救毒株对Vero细胞嗜性的差异。结果成功构建了Vero细胞嗜性差异的CV-A10毒株2种感染性克隆,并在RD细胞内完成病毒拯救,序列测定结果表明拯救毒株与亲本毒株序列一致。拯救毒株rCV-A10-010195能够有效感染Vero细胞并形成典型的细胞病变(cytopathic effect,CPE),而rCV-A10-3482无法有效感染Vero细胞。结论建立了2株Vero细胞嗜性差异的CV-A10毒株反向遗传操作系统,为CV-A10毒株细胞嗜性的研究奠定了基础。Objective To construct infective clones using two Coxsackievirus A10(CV-A10)strains with different cell tropism to Vero cells and rhabdomyosarcoma(RD)as templates respectively and accomplish virus rescue,so as to lay a foundation of the investigation of cell tropism of CV-A10 strain.Methods The genomic sequences of CV-A10 strains with cell tropism differences of Vero^(+)/RD+and Vero^(-)/RD+were amplified respectively and introduced with T7 promoter sequence at the 5′UTR as well as polyA tail and NotⅠrestriction site at the 3′UTR.The obtained genome sequences were connected with linearized pBR322 vector by seamless cloning to obtain full-length plasmids,which were then linearized and cotransfected with T7-RNA polymerase expression plasmids to RD cells to rescue the viruses.The differences of cell tropism of the rescued virus strains to Vero cells were compared by observing cytopathic effect(CPE)and indirect immunofluorescence assay(IFA)after virus infection.Results Two infective clones of CV-A10 strains with different Vero cell tropism were successfully constructed,which were rescued in RD cells.Sequencing results showed that the sequences of rescued strains were consistent with those of parent strains.The rescued strain rCV-A10-010195 effectively infected Vero cells and formed typical CPE,while rCV-A10-3482 did not.Conclusion The reverse genetic operating system of two CV-A10 strains with different cell tropism to Vero cell was developed,which laid a foundation of the investigation of cell tropism of CV-A10 strain.

关 键 词：VERO细胞 柯萨奇病毒A10型 感染性cDNA 病毒拯救 细胞嗜性 

分 类 号：Q75[生物学—分子生物学]