猪繁殖与呼吸综合征病毒E＋M＋N基因重组腺病毒的构建

Construction of recombinant adenovirus co-expressing E, M and N gene of porcine reproductive and respiratory syndrome virus

猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)俗称猪蓝耳病,是由病毒引起的一种高度接触性传染病,可导致母猪晚期流产、早产、死胎、木乃伊胎等繁殖障碍和仔猪严重的呼吸道症状及高死亡率。该病毒已在全世界范围内广泛流行,给养殖业造成了重大的经济损失。目前,传统的疫苗无法提供完全的保护作用,开发研制新型疫苗已迫在眉睫。PRRSV基因组长约15kb,有8个不同程度的开放阅读框(ORFS),即ORF1-ORF7。该病毒具有3个重要的结构蛋白,即E蛋白,M蛋白和N蛋白,这三种蛋白分别由病毒基因ORF5,ORF6和ORF7编码。本试验构建了含猪繁殖与呼吸综合征病毒E+M+N基因的重组腺病毒。首先用反转录聚合酶链反应(RT-PCR)的方法分别扩增出猪繁殖与呼吸综合征病毒E基因、M基因和N基因,将三者用口蹄疫病毒2A序列串联起来,将连接好的E+M+N基因插入腺病毒穿梭载体pAdTrack-CMV。将获得的重组穿梭质粒和腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183内进行同源重组,获得了插有外源基因的重组腺病毒质粒pAd/E+M+N;最后,经PacⅠ酶切线性化后转染HEK-293细胞,成功地得到含有E+M+N基因的重组腺病毒,为重组腺病毒活载体疫苗的研究奠定基础。

In this study,the recombinant adenovirus containing the E,M and N genes simultaneously was successfully obtained for the first time.To construct recombinant adenovirus,the E,M and N were amplified by RT-PCR firstly,then linked by FMDV 2A sequence and inserted into shuttle vector pAdTrack-CMV,being screened and obtained a recombinant plasmid of pAdTrack-CMV/E＋M＋N.A recombinant plasmid of pAdTrack-CMV/E＋M＋N was obtained by screening.The pAdTrack-CMV/E＋M＋N and the pAdEasy-1 system were efficiently constructed into E.coli strain BJ5183 by employing homologous recombination.Exogenous PRRSV genes of recombinant adenovirus pAd/E＋M＋N were born.Finally,the recombinant vector construct was cleaved with PacI,and transfected into HEK-293 packaging cell line.The strain of recombinant adenovirus containing E,M and N gene from PRRSV strain was successfully obtained,thus providing a basis for the research of recombinant replication-defective adenovirus vaccine co-expressing the EP,MP and NP of PRRSV.