两种启动子拯救猪繁殖与呼吸综合征病毒ZJ-JX-2015株效率的比较研究

Comparative study on the efficiency of two promoters to rescue porcine reproductive and respiratory syndrome virus strain ZJ-JX-2015

为比较不同启动子驱动的猪繁殖与呼吸综合征病毒(PRRSV)强毒株ZJ-JX-2015感染性克隆拯救的各重组病毒的效率,本研究将ZJ-JX-2015株全基因组引入无义突变位点(C-G,形成Bst B I酶切位点)作为后期鉴定标签并分成4段经PCR扩增后,分别克隆至含CMV启动子的p SMART-BAC载体和含T7启动子的p WSK-29载体中构建感染性克隆质粒p SMART-BAC-CMV-r PRRSV和p WSK-29-T7-r PRRSV,经PCR及测序鉴定正确后将前者分别转染BHK-21和293T细胞;将后者转染BHK-21(T7)细胞。48 h后收集上述各细胞上清液接种Marc-145细胞并连续传10代。通过观察细胞病变(CPE),收集不同代次Marc-145细胞上清液经PCR及间接免疫荧光试验(IFA)鉴定拯救的各重组病毒。将拯救的各重组病毒以MOI 0.1接种Marc-145细胞,通过测定不同时间各病毒上清液的病毒效价(TCID50)绘制生长曲线。结果显示,各细胞上清液[BHK-21、293T细胞及BHK-21细胞(T7)]在Marc-145细胞中传至第2代时均出现CPE;PCR及测序鉴定结果显示,各代次的各组Marc-145细胞上清液均能扩增到544 bp的目的片段,且均含遗传标记Bst B I酶切位点。IFA结果显示,上述各代次各组Marc-145细胞上清液接种的各细胞中均出现绿色荧光,对照Marc-145细胞无绿色荧光。上述结果表明,拯救了3株重组PRRSV:BHK-r JX15、293T-r JX15(分别转染BHK21及293T细胞所得)及T7-r JX15株[转染BHK-21(T7)细胞所得],且各病毒的遗传稳定性较强。测定各病毒TCID50并绘制各病毒生长曲线,结果显示,在各病毒的感染后期(感染后72 h),BHK-r JX15株的病毒效价显著高于亲本病毒(P<0.05),极显著高于T7-r JX15及293T-r JX15株(P<0.01);293T-r JX15的病毒效价极显著高于T7-r JX15株(P<0.01),但这两株病毒的效价又极显著低于亲本病毒(P<0.001)。且4株病毒的生长趋势基本一致。上述结果表明CMV启动子驱动的重组PRRSV(BHK-rJX15和293T-r JX15)复制水平高于T7启动子驱动的重组病毒(T7-rJX15),尤以CMV启动子驱动并通过BHK细胞拯救的重组病毒(BHK-rJX15)复制水平最高。提示,CMV启动子更适用于构建PRRSV的反向遗传操作系统。本研究为构建PRRSV反向遗传操作系统及其蛋白功能研究、新型疫苗的研发奠定了基础。

To construct a reverse operating system for porcine reproductive and respiratory syndrome virus(PRRSV)strong strain ZJ-JX-2015 driven by two different promoters,the full-length genome of ZJ-JX-2015 was divided into four fragments and the restriction enzyme Bst B I site was introduced by synonymous mutation(C-G)as the genetic marker.Then the homologous recombination method was used to construct the pSMART-BAC vector containing CMV promoter and the pWSK-29 vector containing T7 promoter,which were named as pSMART-BAC-CMV-rPRRSV and pWSK-29-T7-rPRRSV,respectively.After PCR amplification and sequencing,pSMART-BAC-CMV-rPRRSV was transfected into BHK-21 and 293T cells,respectively.while p WSK-29-T7-rPRRSV was transfected into BHK-21(T7)cells.After 48 hours,the supernatant of the transfected cells was collected and inoculated with Marc-145 cells for 10 successive passage.The supernatant collected from the inoculated Marc-145cells with cytopathogenic effect(CPE),were subjected to PCR amplification and sequencing.The rescued recombinant viruses were then inoculated into Marc-145 cells with MOI 0.1,and the virus titer(TCID50)was determined and the growth curve was drawn.The results showed that all cell supernatant(BHK-21,293T cells and BHK-21(T7)cells)showed CPE when transferred to Marc-145 cells in the second passage.The results of PCR and sequencing showed that a 544 bp-target fragment could be amplified from the supernatant,and all of them had the genetic marker Bst B I.IFA results showed that cells inoculated with the supernatant of each generation showed positive signal,while the Marc-145 cells showed no green fluorescence.These results showed that the three recombinant PRRSV strains BHK-r JX15,293T-RJX15(transfected BHK21 and 293T cells,respectively)and T7-RJX15(transfected BHK-21(T7)cells)were rescued,with high genetic stability.The results of the growth curve showed that the titer of BHK-r JX15 was significantly higher than that of the parental virus(P<0.05),and significantly higher than that of T7-rJX15 and293T-rJX15(P<0.01)at the late stage of infection(72 hours after infection).The virus titer of 293T-rJX15 was significantly higher than that of T7-rJX15(P<0.01),but the titer of these two strains was significantly lower than that of the parental virus(P<0.001).The results of one-step growth curve showed that the growth kinetics of the four rescued viruses was basically the same.Furthermore,the replication level of CMV promoter-driven recombinant PRRSV(BHK-rJX15 and 293T-rJX15)was higher than that of the T7 promoter-driven recombinant PRRSV(T7-rJX15).In particular,the rescued virus(BHK-rJX15)had the highest replication capacity.It also showed that CMV promoter is superior to T7 promoter when constructing reverse genetic operating system of PRRSV.This study laid a foundation for further research on PRRSV reverse genetic operating system,protein function,genetic variation,and the development of novel vaccines.