C基因沉默的小反刍兽疫病毒Nigeria 75/1株反向遗传学系统的建立  

Establishment of Reverse Genetics for C-gene Silencing of Peste Des Petits Ruminants Virus Nigeria75/1

作　　者：李菊 石晓玲 杨晓莉 杨东亮 毕冬琳 刘方程 张潇文 李琼毅 柏家林[1,2,3] LI Ju;SHI Xiaoling;YANG Xiaoli;YANG Dongliang;BI Donglin;LIU Fangcheng;ZHANG Xiaowen;LI Qiongyi;BAI Jialin(Engineering Research Center for Key Technology and Industrialization of Cell-based Vaccine,Ministry of Education,P.R China,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China;Key Laboratory of Bioengineering and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;School of Chemical and Biological Engineering,Lanzhou Jiaotong University,Lanzhou 730070,China)

机构地区：[1]西北民族大学,细胞基质疫苗关键技术与产业化教育部工程研究中心,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730030 [3]西北民族大学,生物医学研究中心生物工程与技术国家民委重点实验室,兰州730030 [4]兰州交通大学生物化工学院,兰州730070

出　　处：《病毒学报》2024年第3期563-573,共11页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31860696),题目:PPRV非结构蛋白C抑制RLRs介导的I型IFN信号通路的分子机理研究;兰州市人才创业创新(项目号:2020-RC85),题目:运用CRISPR/Cas9介导的基因打靶技术构建定点敲入siat7e基因的悬浮培养VERO细胞;西北民族大学中央高校基本科研业务费专项(项目号:31920230001),题目:西北民族大学服务学科建设科研平台研究能力提升专项—生物工程学科科研平台群建设项目。

摘　　要：为建立小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)反向遗传学体系,本研究以PPRV Nigeria75/1疫苗株基因组序列为基础,PCR分7个片段扩增病毒基因组,运用重叠延伸PCR(Gene splicing by overlap extension PCR,SOE-PCR)在3’端引入锤头状核酶(Hammerhead ribozyme,HamRz)、5’端引入丁型肝炎病毒核酶(Hepatitis D virus ribozyme,HdvRz)和T7终止子,构建PPRV全长cDNA感染性克隆质粒pBluescript SK-PPRV,进一步构建C基因沉默的病毒全长cDNA感染性克隆染质粒pBluescript SK-PPRVΔC。pBluescript SK-PPRVΔC、pcDNA3.1-N、pcDNA3.1-P和pcDNA3.1-L共转染BSR T7/5细胞,48 hpt后收集细胞反复冻融上清液感染山羊子宫内膜上皮细胞(GEECs),盲传3代,拯救重组病毒命名为rPPRVΔC。重组病毒感染GEECs细胞中RT-PCR扩增出病毒N、P和部分L基因(L2)片段,Western blot检测到N蛋白表达、C蛋白未表达,IFA也检测到N蛋白表达,成功建立了PPRV Nigeria75/1株反向遗传学体系。研究结果为PPRV新型标记疫苗的研制及C蛋白致病机理研究奠定了基础。We wished to undertake reverse genetics of the Peste des petits ruminants virus(PPRV).This study focused on the genome sequence of the PPRV Nigeria75/1 vaccine strain.Seven viral genome fragments were obtained by polymerase chain reaction(PCR)amplification.We introduced a Hammerhead ribozyme at the 3’end.Then,a hepatitis D virus ribozyme and terminator of T7 promoter at the 5’end was introduced using gene splicing by overlap extension PCR.Hence,the plasmid pBluescript SK-PPRV was constructed.Furthermore,we constructed a full-length cDNA infectious cloning plasmid pBluescript SK-PPRVΔC with C-gene silencing.BSR T7/5 cells were co-transfected with pBluescript SK-PPRVΔC and helper plasmids pcDNA3.1-N,pcDNA3.1-P and pcDNA3.1-L.After 48 hpt,cells were collected and the freeze–thaw supernatant of cells was used to infect goat endometrial epithelial cells(GEECs).After blind passaging for three generations,the rescue recombinant virus was named rPPRVΔC.In GEECs infected with recombinant virus,the N,P and partial L2 fragment were amplified by reverse transcription-PCR.Western blotting was employed to detect expression of N protein,but expression of C protein was not detected.An indirect immunofluorescence assay was used to detect N-protein expression.These findings showed that the reverse genetics of PPRV Nigeria75/1 had been established.Our results lay a foundation for the development of a novel PPRV marker vaccine and study of the pathogenesis of C protein.

关 键 词：PPRV Nigeria 75/1疫苗株 C基因 SOE-PCR 病毒拯救 反向遗传学 

分 类 号：S855.3[农业科学—临床兽医学]