人B淋巴细胞刺激因子的真核表达以及表达条件的筛选

Eukaryotic Expression of HsBLyS and Screening of Expressing Conditions

采用基因克隆、重组等方法 ,将人B淋巴细胞刺激因子 (hsBLyS)基因片段从人胎盘cDNA文库中克隆出来 ,测序鉴定后重组构建成真核表达载体pcDNA3.1(+) /hsBLyS .然后用磷酸钙法和脂质体法分别转染真核宿主细胞COS 7,并在其中表达 48h、36h、72h、96h ;表达细胞经超声处理后离心 ,上清进行SDS PAGE鉴定 .结果表明 :对于hsBLyS来说 ,磷酸钙法比脂质体法转染效果好 ,而且对于磷酸钙法 ,表达量是 72h达到最高 .

The full length cDNA of human B lymphocyte stimulator (hBLyS) was amplified by using PCR method from cDNA library of human placenta.After purifying and sequencing,the DNA fragment of functional domain of hBLyS (hsDNA fragment) was amplified by using nest-PCR method from the PCR product.The eukaryotic expression plasmid pcDNA3.1(+)/hsBLyS was constructed with recombinant DNA techniques after purifying and identifying the hsDNA fragment.Then the plasmid pcDNA3.1(+)/hsBLyS was transformed into COS*.7 cells with the calcium phosphate method and lipofection method.The recombination protein was found to be expressed and the calcium phosphate method was better than lipofection method.The best expression time in the calcium phosphate method was 72*#h.