SV40LT抗原基因逆转录病毒载体的构建及其在肝细胞中的表达

Construction of retroviral vector inserted SV40 large T antigen gene and its expression in rat hepatocytes

目的 构建含SV 40LT抗原基因的逆转录病毒载体并转染鼠肝细胞 ,检测SV 40LT抗原基因在肝细胞中的表达情况 ,为肝细胞移植研究打下基础。方法 利用体外基因重组技术构建含SV 40LT抗原基因的逆转录病毒载体并酶切、测序鉴定 ,脂质体介导转染PA3 17细胞 ,G 418抗性筛选阳性克隆 ,通过NIH 3T3细胞测定病毒滴度 ;分离、纯化大鼠肝细胞后 ,将含SV 40LT抗原基因的假病毒颗粒感染肝细胞 ,用PCR及免疫组化法检测转染肝细胞中SV40LT抗原基因的表达情况。结果 ( 1)酶切分析、测序证明重组逆转录病毒载体含有SV 40LT抗原基因 ;( 2 )病毒滴度为 1.3× 10 6 cfu/ml ;( 3 )转染后的肝细胞含有SV40LT抗原基因 ,其表达在转染后 2 4h明显高于 96h(P <0 .0 5 )。结论 成功构建含SV40LT抗原基因的逆转录病毒载体 ,转染后的肝细胞表达有目的基因 。

Objective To construct the retroviral vector inserted SV40 large T antigen gene and transfect it into rat hepatocytes, analyze the status of SV40 large T antigen gene expression in rat hepatocytes, and to establish important basis for clinical hepatocytes transplanation. Methods Retroviral vector inserted SV40 large T antigen gene was(constructed) by DNA recombinant techniques in vitro, then the combinant vector was determined with enzyme(digestion) and sequencing and was transfected into the PA317 cell lines by liposome mediation and screened(anti-G4)18 positive clones. The viral titer was determined with the NIH3T3. After transfected into separated and purified rat primary hepatocytes, the SV40 large T antigen gene expression was detected by PCR and immunohistochemical (methods). Results (1)SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation. (2)The titer of pseudovirion packed by PA317 cell lines was 1.3×10~6CFU/ml. (3)SV40LT antigen gene was(integrated) into rat primary hepatocytes and its expression in transfected cells at 24 hour was higher than 96 hour (P<0.05). Conclusions The retroviral vector inserted SV40LT antigen gene can be constructed successfully and the SV40LT antigen gene can be transfected into hepatocytes and expressed in hepatocytes.