人碱性成纤维细胞生长因子在昆虫细胞sf9中的表达

Expression of Human Basic Fibroblast Growth Factor Gene in sf9 Insect Cell

将人碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)基因cDNA克隆到pFastBacI转移载体中 ,重组质粒转化到感受态细胞DH1 0Bac中 ,使目的基因转座入BacmidDNA中 ,纯化DNA后 ,利用病毒噬斑实验筛选重组的杆状病毒。重组杆状病毒感染Sf9细胞 ,在Sf9细胞中进行表达。Western印迹结果表明 :表达产物为相对分子质量约 1 70 0 0的蛋白 ,与预计的重组蛋白相对分子质量相同 ,并具有较好的免疫学活性。MTT检测证实表达产物能明显促进人脐静脉内皮细胞 (HUVEC)的分裂增生 。

The cDNA encoding human basic fibroblast growth factor(bFGF) was cloned into a transfer factor pFastBac1. After transformation, it was introduced into E.coli DH10Bac. By site-specific transposition, hbFGF gene was intergrated into Bacmid DNA. The isolated recombinant Bacmid DNA were transfected into sf9 insect cells and baculovirus stocks were harvested from the transfected cells. The expressed recombinant hbFGF proteins with the molecular weight of 17 000 were identified by Western blot analysis. MTT results show that rhbFGF proteins have bioactivity to stimulated HUVEC proliferation.