截断型人酸性成纤维细胞生长因子的表达及活性鉴定

目的:稳定且具有活性的截断型人酸性成纤维细胞生长因子(shaFGF)的构建、表达及纯化.方法:以含全长人酸性成纤维细胞生长因子cDNA的质粒pUC-haFGF为模板,设计一对引物,PCR扩增获得shaFGF cDNA,将其克隆到表达载体pET3c中,重组质粒转化大肠杆菌BL21(DE3),0.5 mmol/L IPTG诱导表达,通过离子交换和肝素亲和层析纯化目标蛋白,MTT法检测促分裂活性.结果:shaFGF的表达量约为25%,纯化的shaFGF的促分裂活性与haFGF标准品相当.结论:BL21(DE3)/pET3c表达系统能高效表达shaFGF,纯化得到的shaFGF可供下一步的药学研究.

Expression and Activity Identification of Shortened Human Acidic Fibroblast Growth Factor

AIM: To construct,overexpress and purify a stable and active form of shortened human acidic fibroblast growth factor (shaFGF) in Escherichia coli METHOD: Using the plasmid pUC-haFGF containing full-length human acidic fibroblast growth factor cDNA as a template and a pair of new designed primers,shaFGF cDNA was amplified by Polymerase chain reaction (PCR) The target cDNA fragment was cloned into the expression vector pET3c and expressed in BL21(DE3) by induction The expressed shaFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate The mitogenic activity was assayed by MTT method RESULT:The expression level of shaFGF in Escherichia coli was up to 25% of the total cellular protein The appreciable mitogenic activity of the purified shaFGF was comparable to that of the haFGF standard CONCLUSION:The shaFGF could be expressed at high level in BL21(DE3)/ pET3c expression system The purified recombinant shaFGF was prepared and sufficient for the following pharmaceutical study