肿瘤坏死因子凋亡相关配体基因的克隆和表达

Cloning and expression of human TRAIL gene

目的 :克隆、表达和鉴定肿瘤坏死因子凋亡相关配体 (TRAIL) .方法 :从培养的人外周血淋巴细胞中提取总RNA ,经RT PCR获得TRAIL基因 .将该基因克隆到pGEM Teasy载体中 ,测序鉴定 .将TRAIL基因插入pBV2 2 0表达载体中 ,4 2℃诱导表达 4～ 5h ,进行SDS PAGE分析 .免疫印迹法鉴定TRAIL蛋白的表达 .结果 :DNA测序证明 ,获得了TRAIL基因 ,其序列与GenBank中报道序列完全一致 .SDS PAGE分析表明 ,TRAIL蛋白获得高效表达 ,分子质量为 17ku ,表达量约占菌体总蛋白的 30 % .免疫印迹法鉴定显示 ,该蛋白可与鼠抗人TRAILmAb产生阳性反应 .结论

AIM: To clone, express and identify human TRAIL gene. METHODS: Total RNA was extracted from human PBMC, and the whole length of TRAIL gene was obtained by RT PCR. The TRAIL gene was cloned into pGEM Teasy vector and sequenced. Then the gene was inserted into Eco RⅠ and Bam HⅠ site of pBV220 expression vector. After the recombinant bacteria was induced at 42℃ for 4-5 h,the expressed protein was analyzed by SDS PAGE and western blot. RESULTS: DNA sequencing result showed that TRAIL gene was exactly consistent with the sequence reported in GenBank. SDS PAGE analysis demonstrated that TRAIL protein was expressed in E.coli , and the molecular mass of it is 17 ku. The protein band amounted to 30% of total bacteria protein and could react with anti TRAIL antibody. CONCLUSION: TRAIL gene was successfully cloned and expressed.