麻疹疫苗株Schwarz反向遗传系统的建立  

Construction of a reverse-genetic system of measles vaccine virus Schwarz strain

作　　者：陈宗香[1] 张勇侠[1] 高雅丽 康庄[1] 罗心梅 丛聪[1] 李立[1] 刘兰军[1] CHEN Zong-xiang;ZHANG Yong-xia;GAO Ya-li;KANG Zhuang;LUO Xin-mei;CONG Cong;LI Li;LIU Lan-jun(Chengdu Institute of Biological Products Co.,Ltd.,Chengdu 610023,Sichuan)

机构地区：[1]成都生物制品研究所有限责任公司,四川成都610023

出　　处：《中国生物制品学杂志》2021年第6期637-642,共6页Chinese Journal of Biologicals

摘　　要：目的建立麻疹疫苗株Schwarz(MVSW)反向遗传系统。方法将MVSW全长cDNA分为F1~F7共7个片段,在F1片段的5′端插入T7启动子及锤头核酸酶序列(hammerhead ribozyme,HamRz),F7片段的3′端插入T7终止子及丁型肝炎病毒核酶序列(hepatitis D virus ribozyme,HdvRz),分别将F1~F7片段克隆至pT7-MCS2载体上,7个重组质粒经酶切按顺序拼接MVSW全长cDNA,获得全长质粒pT7MVSW。同时构建表达MVSW核蛋白(N)、磷蛋白(P)、大蛋白(L)的3个辅助质粒。将全长质粒与3个辅助质粒及表达T7RNA聚合酶的质粒(pT7-T7RNAP及pCDIBP-T7RNAP)共同电转染Vero细胞,拯救获得病毒rMV_(SW)。收获的rMV_(SW)在Vero及CEC细胞中进行传代培养,观察细胞病变、检测病毒滴度并进行病毒测序,同时免疫小鼠检测抗r MVSW中和抗体。结果经酶切鉴定,全长质粒及辅助质粒均构建正确。拯救病毒rMV_(SW)在Vero及CEC细胞培养的病变典型特征类似;P2代病毒rMV_(SW)扩增片段与疫苗株MVSW理论序列一致;rMV_(SW)经Vero细胞连续培养P2~P10代病毒滴度稳定在5.5~6.5 lgCCID_(50)/mL之间;rMV_(SW)及疫苗株MVS191诱导的抗rMV_(SW)抗体滴度接近。结论成功建立了麻疹疫苗株MVSW的反向遗传系统,获得的具有良好遗传稳定性及免疫原性的拯救病毒rMV_(SW)有作为疫苗生产用毒种的潜力。Objective To construct a reverse-genetic system for measles vaccine virus Schwarz strain(MVSW).Methods The full-length cDNA sequence of Schwarz strain of measles vaccine virus was analyzed and divided into seven fragments(F1~F7)based on unique restriction enzyme cleavage sites.T7 promoter and hammerhead ribozyme(HamRz)sequence were inserted to the 5′-terminus of fragment F1,while T7 terminator and hepatitis D virus ribozyme(HdvRz)sequence to the 3′-terminus of F7.The seven fragments were synthesized and inserted into vector pT7-MCS2 respectively,then digested with retraction enzyme and spliced in turn to construct full-length plasmid pT7 MVSW.Meanwhile,three helper plasmids coding for nucleoprotein(N),phosphoprotein(P)and large(L)protein of MVSW were constructed.Rescued virus rMV_(SW)was obtained by transfection of Vero cells with the three helper plasmids as well as plasmids pT7-T7RNAP and pCDIBP-T7RNAP for expression of T7RNA polymerase by electroporation.The obtained rescued virus rMV_(SW) was subcultured in Vero cells and primary chick embryo cells(CEC),observed for CPE,determined for titer and sequenced,with which mice were immunized and determined for neutralizing antibody.Results Restriction analysis proved that full-length and helper plasmids were constructed correctly.The typical cytopathic effect(CPE)characteristics of rMV_(SW) in Vero cells were similar to that in CEC cells.The sequence of PCR product of rMVsw of passage 2 was consistent with the theoretical sequence of the vaccine virus strain Schwarz.The titers rMV_(SW) subcultured in Vero cells for 2~10 passages were 5.5~6.5 lgCCID_(50)/mL.However,the antibody titer induced by rMV_(SW) was close to that by vaccine strain MVS191.Conclusion The reverse-genetic system of measles vaccine virus Schwarz strain was successfully constructed,and the rescued virus rMV_(SW)with good genetic stability and immunogenicity was obtained,which might be used as a potential virus seed for vaccine production.

关 键 词：麻疹病毒 疫苗株Schwarz 病毒拯救 反向遗传系统 

分 类 号：R373.16[医药卫生—病原生物学]