用于DNA转染的PRRSV全长感染性cDNA克隆改建及应用  

Development of An Improved DNA-launched Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics System

作　　者：刘长龙[1] 李燕华[1] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所,猪呼吸系统疾病研究室,上海200241

出　　处：《安徽农业科学》2010年第23期12891-12894,12897,共5页Journal of Anhui Agricultural Sciences

基　　金："十一五"支撑项目课题(2006BAD06A01)

摘　　要：[目的]利用核酶自我剪切功能使PRRSV的基因组获得1个精确的基因组3′末端,提高全长感染性cDNA克隆的病毒拯救效率。[方法]用3步PCR方法将获得含有丁型肝炎病毒核酶序列的片段并将其克隆到PRRSV全长cDNA克隆pAPRRS的poly(A)下游,再通过酶切,连接将牛生长激素多聚腺甘酸转录终止序列插入到核酶序列后,构建成全长感染性cDNA克隆pAPRRS-HB,新构建的克隆转染MARC-145细胞,72h后用间接免疫荧光检测病毒N蛋白和非结构蛋白2(nsp2)的表达,并检测细胞上清中病毒的滴度。[结果]成功构建了含有核酶序列的PRRSV基因组全长感染性cDNA克隆pAPRRS-HB。新构建的全长感染性cDNA克隆能够较好地拯救出病毒,拯救效率比pAPRRS提高10倍左右。[结论]该结果为研究PRRSV基因结构与功能奠定了基础。[Objective] The HDV ribozyme （HdvRz） was introduced into immediately downstream genome of the porcine reproductive and respiratory syndrome virus. The infectious cDNA clone with the HdvRz could improve the virus replication efficiency. [Method] Using the PCR,we engineered an HdvRz followed by the bovine growth hormone polyadenylation sequence （BGH） at the 3＇ end of original cDNA clone （pAPRRS）,resulting in pAPRRS-HB. The pAPRRS and pAPRRS-HB were transfected into MARC-145. And the N and nsp2 protein were determined by indirect immunofluorescence assay. The virus titer was tested using TCID50 assay. [Result] Transfection of MARC-145 cells with either pAPRRS or pAPRRS-HB revealed IFA-positive cells,indicating that both infectious clones could rescue the PRRSV. Moreover,more IFA-positive cells were observed in transfections with the HdvRz-containing clone than in transfections with the non-HdvRz-containing clone,suggesting that the HdvRz at 3＇ end of the infectous cDNA clone improves replication efficiency. The rescued efficiency of PRRSV with this system was approximately 10-fold higher than the traditional DNA-launched system without the engineered ribozyme elements,as determined by the virus titer of the recovery virus level in transfected MARC-145 cells. [Conclusion] The results laid a foundation for studying the structure and function of PRRSV gene.

关 键 词：猪繁殖与呼吸综合征病毒 全长感染性cDNA克隆 HDV核酶 DNA转染 

分 类 号：S858.28[农业科学—临床兽医学]