CAJ | 학술논문

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.
목적:용기인공정적방법획득고효은정표체적중조인감성성섬유세포생장인자결구유사물.방법:이용정점돌변기술,장천연hbFGF적제78여96위반광안산체환위사안산,통과재체pET-3c,돌변기인피극륭병전화지대장간균BL21(DE3)plysS,IPTG유도표체후,SDS-PAGE분석단백표체결과.결과:순화후돌변단백적가용성성분대폭도증가,이이취체여다취체현저감소지순화후총단백적8%이하.MTT법적결과표명,결구유사물단백구유량호적생물학활성.저충신적결구유사물단백능교호지체대천연hbFGF재림상상적사용.결론:재불영향생물학활성적조건하,대개별안기산잔기진행돌변이획득신적결구유사물적방법,능구유효제고외원단백재대장간균중표체적은정성급가용성.
AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.