摘要
构建表达HP-PRRSV河南分离株HN1N蛋白基因的siRNA重组伪狂犬病病毒3株,将其感染PK-15细胞,在细胞水平上评价重组伪狂犬病病毒介导的siRNA对N蛋白表达的抑制效果。PCR扩增带CMV-siRNA-poly(A)片段和新霉素基因,通过酶切将CMV-siRNA-poly(A)片段和新霉素基因分别插入伪狂犬病病毒转移载体pUSKBH中,构建重组伪狂犬病病毒通用转移载体pUsiRNA。分别用BamHⅠ和HindⅢ酶切pUsiRNA,回收后与HPPRRSV河南分离株HN1N蛋白基因的3个siRNA分子连接,构建表达N蛋白基因siRNA的重组伪狂犬病病毒表达载体。将鉴定正确的重组伪狂犬病病毒表达载体与伪狂犬病病毒DNA共转染PK-15细胞,经同源重组及噬斑纯化获得表达N蛋白基因siRNA的重组伪狂犬病病毒株,应用PCR及Southern blot鉴定重组病毒。将HN1株N蛋白基因的真核表达质粒pAcGFP1-N转染PK-15,经筛选获得稳定表达N蛋白基因的细胞。将鉴定正确的表达N蛋白基因siRNA的重组伪狂犬病病毒接种于稳定表达N蛋白基因的细胞,利用绿色荧光蛋白基因及半定量RT-PCR检测重组伪狂犬病病毒介导的N蛋白基因siRNA对N蛋白表达的抑制效果。结果显示,PCR扩增到的CMV-siRNA-poly(A)片段约为0.7kb,新霉素基因约1.5kb。经酶切、PCR及测序鉴定,构建了表达siRNA的通用伪狂犬病病毒转移载体pUsiRNA。经PCR及Southern blot检测,获得了3株表达HP-PRRSV河南分离株HN1N蛋白基因siRNA的重组伪狂犬病病毒gG-/siRNAN1、gG-/siRNAN2和gG-/siRNAN3;经荧光显微镜观察和半定量RT-PCR检测表明,重组伪狂犬病病毒介导的N蛋白基因siRNA能够显著抑制N蛋白在PK-15细胞中表达,其中gG-/siRNAN2抑制效果最好。这为深入研究N蛋白基因siRNA抑制HP-PRRSV复制奠定基础,并为HP-PRRSV的防制提供新的思路。
Three recombinant pseudorabies viruses mediated siRNA of nucleocapsid protein gene of highly pathogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV） HN1 were constructed,PK-15 was infected with three recombinant pseudorabies viruses to evaluate the effect of recombination pseudorabies virus mediated siRNA on inhibition of nucleocapsid protein gene ex- pression of HP-PRRSV. CMV-siRNA-poly （A） and Neo genes were amplified respectively by PCR. The recombination pseudorabies virus mediated siRNA universal transfer vector pUsiRNA was constructed after CMV-siRNA-poly（A） and Neo fragments were inserted into transfer vector pUSKBH. According to nucleocapsid protein gene sequence of HP-PRRSV HN1,three small interfer-ing RNA（siRNA） oligonucleotides targeting nucleocapsid protein gene were designed and synthesized.After annealing, oligonucleotides were inserted into pUsiRNA, three recombination pseudorabies virus mediated siRNA of nucleocapsid protein gene expression vectors identified by restriction en-donuclease and sequencing were successfully constructed. Three correct expression vectors were cotransfected into PK-15 with pseudorabies virus DNA,respectively, three recombination pseudor-abies viruses were generated by homologous recombination and plaque purification. The recombi-nant pseudorabies viruses named gG-/siRNANI,gG-/siRNAN2 and gG-/siRNAN3 were detected by PCR and Southern blot. Eukaryotic expression vector pAcGFP1-N was transfected into PK-15 by liposome to express nucleocapsid protein stably after selecting with G418. And then the recom- binant pseudorabies viruses gG-/siRNAN1 ,gG-/siRNAN2 and gG-/siRNAN3 infected PK-15 to e- valuate the effect of the virus mediated siRNA on inhibition of nucleocapsid protein gene expres- sion of HP-PRRSV by fluorescence microscope and RT-PCR semi-quantitatively. The results indi-cated that the size of CMV-siRNA-poly（A） was about 0.7 kh and Neo was about 1.5 kb. Three re-combinant pseudorabies viruses mediated nucleoeapsid protein gene siRNA were successfully con-structed,and could significantly inhibit expression of nucleocapsid protein in PK-15. Among of them,the inhibition effect of gG-/siRNAN2 was the best. This study laid a foundation for further studies on inhibition of HP-PRRSV by siRNA of nucleocapsid protein.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第8期1142-1149,1153,共9页
Chinese Journal of Veterinary Science
基金
河南省高校科技创新人才支持计划资助项目(2008HASTIT01)
河南省教育厅自然科学研究计划资助项目(2011A230012)
关键词
高致病性
PRRS
N蛋白基因
SIRNA
重组伪狂犬病病毒
highly pathogenic porcine reproductive and respiratory syndrome
N protein gene
smallinterfering RNA（siRNA）
recombination pseudorabies virus
