猪传染性胃肠炎病毒sM基因反义逆转录病毒的包装和鉴定

THE PACKAGE AND IDENTIFYING OF sM GENE RETROVIRUS VECTOR FOR TGEV

将编码猪传染性胃肠炎病毒(TGEV)sM基因的cDNA反向插入逆转录病毒表达载体pLXSN中,质脂体法将重组质粒plxas-sM转染PA317细胞,经抗生素G418(500μg/mL)筛选出稳定的产毒细胞克隆.分别扩大培养细胞克隆,取其上清液感染小鼠成纤维细胞NIH3T3,测定细胞克隆产生的重组病毒滴度,最高达1.50×105CFU/mL.提取重组病毒的RNA进行RT-PCR分析,结果表明成功获得plxas-sM逆转录病毒.

The cDNA for sM gene full length of TGEV was inserted into retroviral vector pLXSN to get pLXSN-sM, and it was transfected into packaging cells line PA317 with Lipofectamine. The viral supenatants of the clones selected with G418 (500 μg/mL) were detected by murine fibre cell NIH3T3. The result showed that the highest viral titer among the clones was 1.50×10~5 CFU/mL. Total RNA of the viral supenatants, RT-PCR analysis showed that cDNA amplifited had same legth with that of sM gene. It indicated that recombinant retrevirus was obtained.