重组GIP蛋白的原核优化表达及其生物活性的研究  

Optimized Prokaryotic Expression and Bioactivities of The Recombinant Human Glucose-dependent Insulinotropic Polypeptide

作　　者：胡质毅[1] 龙綮新[1] 王珣章[1] 

机构地区：[1]中山大学生命科学学院有害生物控制及资源利用国家重点实验室

出　　处：《生物化学与生物物理进展》2006年第11期1086-1093,共8页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30572453);世界银行贷款资助项目(129A01052);广东省社会发展计划资助项目(2005B20301024).~~

摘　　要：葡萄糖依赖性促胰岛素多肽或抑胃肽(glucose-dependentinsulinotropicpolypeptideorgastricinhibitorypeptide,GIP)是由42个氨基酸组成的胃肠调节肽,在高血糖背景下能够刺激胰岛素释放,能够抑制胃酸分泌、促进神经细胞增生,具有广泛的临床应用价值.化学提取或人工合成GIP,成本过高,不宜规模化生产,故应用基因工程技术研制重组人GIP(rhGIP)并探讨其生物活性有积极的现实意义.人工合成具有大肠杆菌偏爱密码子的编码GIP成熟肽的cDNA序列,利用pET32a(+)系统进行原核表达;在小规模发酵条件下,进行优化诱导表达和目的蛋白的亲和纯化;通过检测SD大鼠胃酸分泌和血糖浓度,对纯化后的rhGIP进行生理活性研究;通过形态学观察和培养基中NO含量测定,检测rhGIP对PC12细胞NO自由基生成量的影响;应用Aβ25-35加入培养基造成PC12细胞神经损伤模型,分别以高、中、低剂量rhGIP作用于此模型,通过MTT(2-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazoliumbromide)法测定PC12细胞的活性.结果显示,成功克隆了人GIP基因,诱导表达的rhGIP占细胞总蛋白质的35%,部分可溶,部分以包涵体形式存在.经过诱导表达的重组蛋白质分子质量约为26ku,与理论值相符.纯化后的rhGIP具有免疫活性.优化诱导表达条件为表达菌生长密度A600值0.50,IPTG浓度0.5mmol/L,温度37℃,诱导表达时间4h.裂解上清液经固定化金属亲和层析一步法层析后,表达的水溶性rhGIP融合蛋白的最后得率为1.2mg/L菌液,纯度为85%.纯化后的rhGIP能够使SD大鼠胃液pH值增高,其抑制胃酸分泌作用与生理盐水对照组比较差异有显著性(P<0.05),而rhGIP组和标准品GIP组比较差异无显著性.在高血糖背景下,注射rhGIP15min后,大鼠血浆血糖浓度较基础血糖显著降低(P<0.05),30min时与单独注射葡萄糖的模型对照组比较,差异无显著性,而rhGIP组和标准品GIP组其差异不显著.在rhGIP对神经细胞的营养和对PCGIP, the acronym from gastric inhibitory peptide or glucose-dependent insulinotropic polypeptide, which is a kind of gastrointestinal regulatory peptide composed of 42 amino acids, plays an important role in stimulating insulin releasing in the pancreas, inhibiting gastric acid secretion in the stomach, as well as inducing progenitor cell proliferation in the brain. The application of GIP is promising in clinic. It is difficult and costly to get GIP with chemical extraction method, and it can＇t be produced cosmically, so it is valuable and significant to produce recombinant GIP by genetics and test its bioactivity. The cDNA sequence coding for human GIP mature peptide and composing of preferred codons of E. coli. was synthesized, and was expressed in prokaryotic expression pET32a （＋） system. The recombinant E. coli was fermented in a small scale and the expression condition was optimized. The expressed recombinant human GIP （rhGIP） was purified by affinity method. The bioactivity of the purified rhGIP was tested through its effect on inhibiting gastric acid secretion and decreasing blood glucose concentration in SD rats. The effect on PC12 cell producing NO free radical was assessed through morphology observation and the NO concentration test. A PC12 cell injury model was constructed by adding Aβ25-35 into the culture medium, different dose of rhGIP was added into this model, and the activity of PC12 was tested by MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. The results suggest that hGIP gene is cloned successfully, the recombinant protein constitutes 35% of the total protein of the cells, some of which was soluble, while the others exist as inclusion body. The relative molecular mass of the recombinant protein is 26 ku as expected. The purified rhGIP showed immunoreactivity. The optimal condition for inducing to expression is with the cell concentration of 0.5 in A600 value, the IPTG concentration of 0.5 mmol/L, under 37℃ and induced for 4 h. The cell lysis

关 键 词：抑胃肽 葡萄糖依赖性促胰岛素多肽 原核表达 PC12细胞 生物活性 

分 类 号：Q81[生物学—生物工程]