高致病性猪繁殖与呼吸综合征病毒反向遗传操作系统的构建

Construction of a reverse genetic system of highly pathogenic porcine reproductive and respiratory syndrome virus

采用分子生物学方法改造pBluescriptⅡSK(+)载体的多克隆位点,并使其获得CMV启动子序列;在获得该病毒的全长cDNA克隆的基础上,采用酶切、连接、转化等方法将该病毒的全长cDNA置于CMV真核启动子序列的下游,获得含有CMV启动子和HP-PRRSV XX-2012株全长cDNA克隆的重组质粒pSK-CMV-XX-2012;最后,将重组质粒直接转染Marc-145宿主细胞,拯救出HP-PRRSV XX-2012株。结果显示,含有病毒全长cDNA克隆的重组质粒直接转染Marc-145后,获得拯救病毒。病毒生长特性试验结果显示,拯救病毒与亲本病毒在Marc-145细胞上具有相似的增殖特性,且拯救病毒序列含有不同于亲本病毒的分子标记(突变产生的MluⅠ酶切位点)。本研究建立了一种研究HP-PRRSV反向遗传操作系统的简单、快速、节约时间和成本的方法。

The multiple cloning sites of pBluescript Ⅱ SK（＋） were recreated, and the sequences of CMV promoter were added in the modified vector. Secondly, the recombinant plasmid pSK-CMV-XX-2012 was constructed by molecular biology technology of enzyme digestion,connection, and transformation. The recombinant vector contains CMV promoter and the downstream full-length cDNA of virus. Terminally, the recombinant plasmid was transfected into host cells of Marc-145 to rescue XX-2012 strain HP-PRRSV. The results showed that the transcription of viral genomic RNA could be synthesized, and the rescued virus could be acquired, after the full-length cDNA clone was directly transfected into Marc-145 cells. The results of virus growth characteristics tests showed that the rescued virus was similar to that of with parental virus in Marc-145 cells,and the rescued virus could be distinguished from the parental with the mutant genetic marker of Mlu I enzyme site. The method which is simple,convenient operation,and saving time and cost was established to obtain reverse genetic system of PRRSV in vitro.