小反刍兽疫病毒C蛋白在外周血单个核细胞中的过表达及鉴定  

Overexpression and identification of peste des petits ruminants virus C protein in peripheral blood mononuclear cells

作　　者：苏乾隆 赵帅阳[1] 蒙学莲[1] 朱学亮[1] 李彦敏 孙跃峰 张志东[1,2] SU Qian-long;ZHAO Shuai-yang;MENG Xue-lian;ZHU Xue-liang;LI Yan-min;SUN Yue-feng;ZHANG Zhi-dong(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]中国农业科学院,兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]西南民族大学畜牧兽医学院,四川成都610041

出　　处：《中国兽医科学》2021年第10期1246-1252,共7页Chinese Veterinary Science

基　　金：“十三五”国家重点研发计划项目(2016YFD0500108);甘肃省科技计划国际科技合作类项目(20YF3WA008)。

摘　　要：为探索小反刍兽疫病毒(peste des petits ruminants virus,PPRV)非结构蛋白C(PPRV-C)的功能及其调节宿主细胞免疫抑制的分子机制,首先根据GenBank中PPRV-C蛋白基因序设计引物,通过PCR扩增获得PPRV-C蛋白基因,将其克隆至慢病毒载体PLVX-IRES-puro上,并命名为PLVX-IRES-puro-PPRV-C,随后连同辅助质粒共同转染HEK-293T细胞,收集上清,浓缩后测定重组慢病毒效价。用包装好的重组慢病毒感染绵羊外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)12 h后,换维持液继续培养,一定时间后收集细胞,检测PPRV-C的mRNA和蛋白表达水平;同时用空载体慢病毒做空白对照。结果显示:成功地构建了PPRV-C蛋白过表达重组慢病毒载体,包装的重组慢病毒效价为(9.6±8.8)×107TU/m L。重组慢病毒感染(MOI=10)后,检测到PPRV-C蛋白mRNA的转录和蛋白的表达结果表明,构建的PPRV-C慢病毒能在PBMCs中高效表达,同时也为今后进一步研究PPRV-C蛋白的功能奠定了基础。In order to explore the function of non-structural protein C(PPRV-C) of peste des petits ruminants virus(PPRV) and its molecular mechanism of regulating the immunosuppression of host cells,the primers were designed according to the gene sequence of PPRV-C protein in Gen Bank,and the PPRV-C protein gene obtained by PCR amplification was cloned into the lentiviral vector PLVX-IRES puro,which was named as PLVX-IRES-puro-PPRV-C.Subsequently,the lentiviral expression plasmid and the helper plasmid were co-transfected into HEK-293 T cells,and the titer of recombinant lentivirus was determined after the supernatant was collected and concentrated.After the peripheral blood mononuclear cells(PBMCs) of sheep were infected with the packaged recombinant lentivirus for 12 h,culture medium was changed for further culture.After a certain period of time,the cells were collected, the mRNA and protein expression levels of PPRV-C were detected,and the empty vector lentivirus was used as blank control simultaneously.The results showed that the recombinant lentiviral vector overexpressing PPRV-C protein was successfully constructed,and the packaged recombinant lentiviral titer was(9.6±8.8)×107 TU/m L. After recombinant lentivirus infection(MOI=10),the mRNA transcription and protein expression of PPRV-C protein were detected in PBMCs.The results indicated that the PPRV-C lentivirus constructed can be efficiently expressed in PBMCs,which laid the foundation for further research on the function of PPRV-C protein in the future simultaneously.

关 键 词：小反刍兽疫病毒 非结构蛋白C 慢病毒 外周血单个核细胞 

分 类 号：S852.659.5[农业科学—基础兽医学]