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