摘要
将编码人 TNFR75的 c RNA与血管内皮细胞特异性启动子 (KDRp)及缺失自身启动子的逆转录病毒载体 p LXSN- D2 99重组 .重组质粒 p LXSN- D2 99- KDRp- TNFR75与脂质体共转染包装细胞 PA31 7,经抗生素 G41 8(60 0 mg/L)筛选 1 4d,获得 1 5个稳定的产病毒细胞克隆 .将各细胞克隆分别扩大培养收集所产病毒上清 ,并感染 NIH3T3细胞检测病毒滴度 ,其中 1个克隆滴度达 2×1 0 5CFU/ml.提取该克隆细胞总 RNA进行 RT- PCR分析 ,获得的 c DNA片段长度与目的基因一致 .结果提示 ,建立了 TNFR75反转录病毒产毒细胞系 .
The cDNA for TNFR75 and the promotor of kinase domain receptor (KDRp) were recombinated with retroviral expression vector pLXSN\|D 299 without promotor.The recombinated plasmid was transfected into packaging cell PA317 using lipofectin method.The transfectants were selected by G418(600 mg/L) for 14 days and 15 stable cell clones grew.Viral supernatants of the clones were checked by NIH 3T3 cells to detect the titer of the virus.The results showed that the highest viral titer among 15 clones was 2×10 5 CFU/ml.Total RNA of the clones with the highest viral titer were extracted.RT\|PCR analysis showed that cDNA amplifited had the same length with that of TNFR75.It indicated that the cell line which could produce virus containing TNFR75 was constructed.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2001年第2期169-172,共4页
Chinese Journal of Biochemistry and Molecular Biology
