人p58.2重组逆转录病毒载体的构建及稳定包装细胞系的鉴定

Construction and identification of recombinant defective retroviral vector with human p58.2 gene and stable virus-producing cell lines

目的 构建并鉴定携带人p5 8 2基因的重组逆转录病毒载体及稳定的包装细胞系。方法 用PCR技术从pSPORT1-p5 8 2扩增出编码p5 8 2N端 1～ 3 45个氨基酸的全长基因 ,定向克隆入逆转录病毒载体pMSCVneo中 ,酶切鉴定 ;脂质体法将重组逆转录病毒转入包装细胞系PT67,G418筛选建立稳定产病毒的细胞株。结果 双酶切鉴定 ,成功构建重组逆转录病毒载体 ;重组逆录病毒载体转入包装细胞 ,经G418筛选 ,形成了抗性克隆 ,并测得病毒滴度为 3×10 5cfu/ml,提示构建携带人p5 8 2基因的逆转录病毒载体及稳定的包装细胞系成功。结论 构建人p5 8 2基因的逆转录病毒载体可行 。

Objective To construct and confirm a recombinant defective retroviral vector carrying Hp58 2 gene and a stable virus-producing packaging cell line. Methods Gene encoded p58 2 was amplified by PCR from pSPORT1-p58 2. The gene was cloned into a retroviral vector pMSCVneo and recombinant vector plasmid DNA was analysed after digested by appropriate restriction enzymes. The recombinant plasmid was then transfered into packaging cell PT 67 with Lipofectin method. Stable virus-produing cell line was obtained by screen of G418. Results The recombined vector pMSCVneo-p58 2 was successfully constructed, and confirmed by enzyme digestion analysis. Anti-G418 positive clones were also achieved with a titer of 3×10 5 cfu/ml. Conclusion Construction of eukaryotic cell expression retroviral vectors is feasible and has the potential to be used in gene therapy of hemalogical disorders by gene therapy.