CAJ | 학술논문

为了降低由人碱性成纤维细胞生长因子(hbFGF)的广谱促分裂活性引起的潜在副作用,用中性氨基酸丙氨酸取代了hbFGF第108位的丝氨酸,构建了促分裂活性降低的hbFGF突变体(mhbFGF).IPTG诱导突变体在大肠杆菌BL21(DE3)中高效表达.mhbFGF的表达量约为全菌体蛋白的30%.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法检测促分裂活性表明,mhbFGF的促分裂活性显著低于野生型hbFGF.纯化的mhbFGF可用于进一步的药效和安全性研究.
위료강저유인감성성섬유세포생장인자(hbFGF)적엄보촉분렬활성인기적잠재부작용,용중성안기산병안산취대료hbFGF제108위적사안산,구건료촉분렬활성강저적hbFGF돌변체(mhbFGF).IPTG유도돌변체재대장간균BL21(DE3)중고효표체.mhbFGF적표체량약위전균체단백적30%.통과리자교환화간소친화층석종균체상청중순화목표단백.MTT법검측촉분렬활성표명,mhbFGF적촉분렬활성현저저우야생형hbFGF.순화적mhbFGF가용우진일보적약효화안전성연구.
In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.