重组人碱性成纤维细胞生长因子突变体[Ser^(69,87)]的表达、纯化及其稳定性研究  

Study on Expression,Purification,and Stability of Recombinant Human Basic Fibroblast Growth Factor Mutant, [Ser^(69,87)] hbFGF

作　　者：吴晓萍[1] 冯辉[1] 李校堃[1] 苏志坚[1] 郑青[1] 黄亚东[1] 

机构地区：[1]暨南大学医药生物技术研究开发中心,广州510632

出　　处：《中国药科大学学报》2005年第2期168-172,共5页Journal of China Pharmaceutical University

基　　金：国家高技术研究发展计划 ("八六三"计划 )资助项目 (No .2 0 0 1AA2 15 13 1) ;国家"十五"重大生物医药专项资助项目(2 0 0 2AA2Z3 3 18)~~

摘　　要：目的:对野生型人碱性成纤维细胞生长因子(hbFGF)进行突变改造以提高其稳定性。方法:采用PCR突变方法,将hbFGFcDNA第6 9位和87位的Cys密码子突变为Ser密码子,所得的突变片断插入表达载体pET3c的表达框架中,构建了表达菌株BL2 1(DE3) /pET3c [Ser69,87]hbFGF ,IPTG诱导表达。通过离子交换、肝素亲和两步层析的方法分离纯化突变体蛋白[Ser69,87]hbFGF。MTT法测定重组蛋白的促分裂活性。并对突变体蛋白的抗蛋白酶水解能力、热稳定性及其酸碱稳定性进行了检测。结果:所构建的表达菌株,其[Ser69,87]hbFGF的表达量占菌体总蛋白的30 %以上。经两步层析纯化,获得了纯度达98%的突变体[Ser69,87]hbFGF。纯化的样品促Balb/c 3T3细胞增殖的活性与野生型hbFGF相当。突变体[Ser69,87]hbFGF抗胰蛋白酶水解的能力较野生型hbFGF强;热稳定性和酸碱稳定性也较野生型hbFGF高。结论:突变体[Ser69,87]hbFGF的生物学活性与野生型hbFGF相当,其稳定性明显高于野生型hbFGF。AIM:To enhance the stability of wide-type human ba sic fibroblast growth factor(hbFGF) by site-directed mutation.METHOD: The codons of the 69 th and the 87 th cysteine of hbFGF cDNA were replaced by serin e codon by application of PCR. The mutant fragment was then cloned in t o the expression vector pET3c.In addition,the expression of [Ser 69,87] hbFGF in BL21(DE3)/ pET3c-[Se r 69,87]hbFGF was induced by IPTG, and the expressed [Ser 69,87]h bFGF was finally purified by ionic exchange and heparin affinity chromatography from the supernatant of the bacteria lysate.The mitogenic activity of the purified sampl e was determined by MTT. The resistant potency to the proteinase,the heat and acid alkali of the mutant were also analyzed.RESULT: The expression level of [Ser 69,87]hbFGF in BL21(DE3) / pET3c-[S er 69,87]hbFGF was up to 30% of the total bacterial protein.The purity o f [Ser 69,87]hbFGF obtained after two-step chromatography was 98%. T he mitogenic activity of the purified [Ser 69,87]hbFGF was comparable to that of the wide-type hbFGF. The anti-enzymolysis capability of [Ser 69 ,87]hbFGF to trypsin was much greater than that of the wide-type hbFGF. Th e heat and acid alkali stabilites of [Ser 69,87]hbFGF at 37 ℃ were also much higher than those of hbFGF. CONCLUSION:While t he biological activity of [Ser 69,87]hbFGF was kept similar to the wide -type hbFGF,the stabi lity of the mutant was remarkably enhanced.

关 键 词：人碱性成纤维细胞生长因子 突变体 稳定性 

分 类 号：Q819[生物学—生物工程]