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目的:用基因工程的方法获得高效稳定表达的重组人碱性成纤维细胞生长因子结构类似物。方法:利用定点突变技术,将天然hbFGF的第78与96位半胱氨酸替换为丝氨酸,通过载体pET-3c ,突变基因被克隆并转化至大肠杆菌BL21( DE3) plysS,IPTG诱导表达后,SDS-PAGE分析蛋白表达结果。结果:纯化后突变蛋白的可溶性成分大幅度增加,而二聚体与多聚体显著减少至纯化后总蛋白的8 %以下。MTT法的结果表明,结构类似物蛋白具有良好的生物学活性。这种新的结构类似物蛋白能较好地替代天然hbFGF在临床上的使用。结论:在不影响生物学活性的条件下,对个别氨基酸残基进行突变以获得新的结构类似物的方法,能够有效提高外源蛋白在大肠杆菌中表达的稳定性及可溶性。
OBJECTIVE: To obtain a highly efficient and stable recombinant human basic fibroblast growth factor structural analog by genetic engineering. Methods: Site-directed mutagenesis was used to replace the cysteines at positions 78 and 96 of native hbFGF with serine. The mutant gene was cloned and transformed into E. coli BL21 (DE3) plysS through vector pET-3c. After IPTG induction, SDS-PAGE analysis of protein expression results. Results: After purification, the soluble components of muteins increased significantly, while the dimers and multimers significantly decreased to below 8% of the total purified proteins. The results of the MTT assay showed that the structural analogue protein has good biological activity. This new structural analogue protein can better replace the clinical use of natural hbFGF. Conclusion: The method of mutating individual amino acid residues to obtain new structural analogs without affecting the biological activity can effectively improve the stability and solubility of the foreign protein expressed in E. coli.