猪生殖与呼吸综合征病毒5′非翻译区序列的反向遗传操作

Reverse genetic manipulation for 5′UTR sequence of porcine reproductive and respiratory syndrome virus

以猪生殖与呼吸综合征病毒(PRRSV)北美株感染性克隆pCBC2为平台进行反向遗传操作,利用突变PCR获得在病毒基因组5′非编码区(5′UTR)和ORF1起始密码子之间插入NdeⅠ的全长克隆pTLNd4,并以此为基础将5′UTR替换为欧洲株的5′UTR,获得全长克隆pTLV8。以体外转录的RNA转染MARC-145细胞。结果显示,pTLNd4产生了细胞病变(CPE),而pTLV8未产生。通过RT-PCR和Northern blot试验,分别对这2株突变病毒的基因组RNA复制和亚基因组mRNA转录水平进行了分析,同时还对pTLNd4进行了病毒学试验。证实,它们与亲本病毒无论从分子生物学水平或病毒学水平都无明显差异;pTLV8虽未产生CPE,但在病毒传代细胞中检测到了基因组RNA,说明替换不同基因型5′UTR的突变病毒虽然丧失了复制能力和感染性,但仍然能够提供病毒RNA合成所需要的顺式调控因子。

Based on infectious full-length cDNA clone pCBC2 of porcine reproductive and respiratory syndrome virus（PRRSV）,pTLNd4 was obtained by PCR-based mutagenesis,in which a Nde Ⅰ was inserted between the end of the 5＇ untranslated region（UTR） and the translation initiation codon of ORF1. Then the 5＇UTR of North American type was substituted with that of European type to obtain chimeric clone pTLVS. The RNAs via in vitro transcription were respectively transcribed into MARC-145 cells for further cytopathic effect（CPE） observation,genomic and subgenomic mRNA analysis. The routine virological experiments were also conducted such as plague morphology to compare this mutant viruses with the wild type. consequently,pTLV8 lost the ability to cause CPE and pTLNd4 was the same as the wild type pCBC2 at a molecular biological level and at a virus biological characteristic level. The results demonstrated that separation of 5＇UTR from ORFs is possible,which provided the foundation for further genetic manipulation in this region,The European genotype UTR could partially function in the background of the North American genotype although,genetically diversified,and the cis-acting elements in the 5＇UTR may need additional cis or trans-acting elements to be fully functional.