重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架，ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌，用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶，后者可催化高水平的ｂＦＧＦ基因表达，其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性，并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。

Overexpression and Characterization of Human Basic Fibroblast Growth Factor from Escherichia coli Cells

The cDNA sequence coding human basic fibroblast growth factor (bFGF) was cloned downstream of the bacteriophage T7 promoter.A upstream short S peptide was used to increase the translation efficiency of bFGF.With induction of IPTG,the recombinant S gene bFGF protein was overexpressed as 42.5% of total bacterial proteins.Heparin sepharose affinity chromatography was used to purify the bFGF fusion protein.Western blot analysis showed that human bFGF specific monoclonal antibody could detect the recombinant bFGF protein.Further experiments demonstrated that the recombinant fusion protein could stimulate the proliferation of the quiescent NR6R 3T3 fibroblasts and the bFGF biological activity could be neutralized by human bFGF specific neutralizing antibodies.