带SV40LT抗原基因的逆转录病毒载体的构建

摘要

Objective: To construct the retroviral vector inserted SV40 large T antigen gene to proliferate the primary hepatocyte. Methods: Retroviral vector inserted SV40 large T antigen gene was constructed by DNA recombinant techniques in vitro. The combinant vector was determined with enzyme digestion and sequencing and transfected into packaging cell lines PA317 by means of liposome - mediated method. After anti - G418 positive clones were screened, the viral titer was determined with the NIH3T3. Results:SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation in success. The titer of pseudovirion packed by PA317 cell lines was 1.3 ×106 CFU/ml. Conclusions:The retroviral vector inserted SV40 large T antigen gene can be constructed successfully and the transfected PA317 cell lines can produce high titer pseudovirion.%目的构建带有SV40LT抗原基因的逆转录病毒载体.方法采用体外重组技术构建带SV40LT抗原基因的逆转录病毒载体,酶切、测序进行鉴定,脂质体介导转染PA317包装细胞,经G418筛选出抗性克隆,通过NIH3T3细胞测定病毒滴度.结果酶切分析、测序证明重组逆转录病毒载体含有SV40LT抗原基因,且为正向插入.NIH3T3细胞测定病毒滴度为1.3×106CFU/ml.结论成功构建了含SV40LT抗原基因的逆转录病毒载体并包装出了高滴度的假病毒颗粒.