CAJ | 학술논문

目的:为了研究缺失核定位信号区的人碱性成纤维细胞生长因子(hbFGF)独特的转运机制,在大肠杆菌BL21(DE3)中高效表达和纯化了缺失核定位信号区的hbFGF.方法:将PCR扩增得到的hbFGF cDNA片断克隆到表达载体pET3c中,重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,通过离子交换和肝素亲和层析从菌体上清中纯化目标蛋白,MTT法检测促分裂活性.结果:hbFGF的表达量约为全菌体蛋白的20%,纯化的缺失核定位信号区的hbFGF的促分裂活性与缺失核定位信号区的hbFGF标准品相当.结论:BL21(DE3)/pET3c表达系统能高效表达缺失核定位信号区的hbFGF,纯化得到的hbFGF可用于进一步的研究.
목적:위료연구결실핵정위신호구적인감성성섬유세포생장인자(hbFGF)독특적전운궤제,재대장간균BL21(DE3)중고효표체화순화료결실핵정위신호구적hbFGF.방법:장PCR확증득도적hbFGF cDNA편단극륭도표체재체pET3c중,중조질립전화대장간균BL21(DE3),IPTG유도표체,통과리자교환화간소친화층석종균체상청중순화목표단백,MTT법검측촉분렬활성.결과:hbFGF적표체량약위전균체단백적20%,순화적결실핵정위신호구적hbFGF적촉분렬활성여결실핵정위신호구적hbFGF표준품상당.결론:BL21(DE3)/pET3c표체계통능고효표체결실핵정위신호구적hbFGF,순화득도적hbFGF가용우진일보적연구.
AIM:To study the mechanism of the unique export of one of human basic fibroblast growth factor (hbFGF) forms lacking the N-terminal nuclear localization signal (NLS),we high expressed and purified this hbFGF form in E.coli strain BL21(DE3).METHODS:The cDNA fragment of the hbFGF amplified by polymerase chain reaction (PCR) was cloned into the expression vector pET3c and expressed in BL21(DE3) by IPTG induction.The expressed hbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate.The mitogenic activity was measured by MTT.RESULTS:The expression level of hbFGF in E.coli was about 20% of the total cellular protein.The appreciable mitogenic activity of the purified hbFGF was comparable to that of hbFGF standard.CONCLUSION:The BL21(DE3)/ pET3c expression system could be used to high express hbFGF lacking NLS.The purified recombinant hbFGF was prepared and sufficient for further study.