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
李菊 1石晓玲 2杨晓莉 3杨东亮 3毕冬琳 3刘方程 1张潇文 1李琼毅 3柏家林3
作者信息
- 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;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
- 2. 兰州交通大学生物化工学院,兰州 730070
- 3. 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;西北民族大学生命科学与工程学院,兰州 730030;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
- 折叠
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-PPRV△C.pBluescript SK-PPRV△C、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rPPRV△C.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.
Abstract
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-PPRV△C with C-gene silencing.BSR T7/5 cells were co-transfected with pBluescript SK-PPRV△C 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 rPPRV△C.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Key words
PPRV Nigeria75/1/C gene/SOE-PCR/Virus rescue/Reverse genetics引用本文复制引用
基金项目
国家自然科学基金(31860696)
兰州市人才创业创新项目(2020-RC85)
中央高校基本科研业务费专项西北民族大学项目(31920230001)
出版年
2024
NETL
NSTL
万方数据