TH基因逆转录病毒表达载体的构建及产病毒细胞系的建立

目的　构建含有鼠酪氨酸羟化酶 (TH)基因的重组逆转录病毒表达载体 p N2 ATH,建立 PA317产病毒细胞系。方法　将质粒 p GEM- 2上酶切下的 THc DNA片段与线性化的逆转录病毒表达载体 N2 A连接 ,克隆出重组逆转录病毒表达载体 p N2 ATH。把 p N2 ATH质粒转入 PA317包装细胞 ,通过 G4 18抗性筛选 ,NIH3T3细胞测定病毒滴度 ,得到高滴度产毒 PA317/p N2 ATH细胞株 ,免疫组化测定细胞 TH表达。结果　重组质粒 p N2 ATH经酶切鉴定证明 THc DNA正向插入 N2 A表达载体中 ,PA317/p N2 ATH产毒细胞高效表达 TH。结论　成功地构建了重组逆转录病毒表达载体p N2 ATH,建立了 PA317/p N2 A产毒细胞株

Construction of a recombinant retroviral expression vector pN2ATH and establishment of producing virus cell line PA317/pN2ATH

Objective To construct a recombinant retroviral vector pNA 2TH encoding mice TH and obtain virus producing cells.Methods The linear vectors N 2A and the THcDNA fragments from plasmids pGEM 2 were conjugated by T4DNA ligase. The recombined retroviral vector pN 2ATH was cloned and identified. The vector pN 2ATH was transferred into PA317 packaging cells by electroporation method. The PA317/pN 2ATH packaging cells were obtained with G418 selective culture, determined its viral titers with NIH 3T 3 cell and detected its expression of TH gene in cells by immunohistochemistry.Results The restriction analysis of pN2ATH with ApaI revealed that the position and size of THcDNA insertion were consistent with the sequence of prediction. After G418 selection, high titer PA317/pN 2ATH cells were obtained and expressed TH successfully.Conclusion The recombinant retroviral vector pN 2ATH is constructed and PA317/pN 2ATH packaging cell line has high viral titer and TH expression.