重组人GIP衍生物的克隆、表达、纯化及活性研究  

Cloning,Expression,Purification and Its Biological Activity Study of a Novel GIP Analog

作　　者：宋文程[1] 金明飞[1,2] 李冬青[1] 赵丽芬[1] 张洪丹[1] 黄静[1] 吴自荣[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062 [2]中国科学院上海巴斯德研究所,上海200025

出　　处：《中国生物工程杂志》2010年第6期38-43,共6页China Biotechnology

基　　金：国家重大新药创制项目(2009ZX09102-253)

摘　　要：葡萄糖依赖性促胰岛素释放肽(glucose-dependent insulinotropic polypeptide,GIP)具有促胰岛素分泌的作用,但因体内半衰期短而限制了其应用。对二肽酶4(dipeptidase IV,DPP IV)识别位点等氨基酸进行改造可有效延长其半衰期。通过人工合成基因或PCR定点突变的方法合成GIP不同突变体基因,将其克隆到大肠杆菌表达载体pET32a(+)中进行诱导表达,通过亲和层析,纯化获得各对应的多肽。经肠激酶酶切后,分别在昆明鼠及糖尿病模型鼠体内研究其生物活性。结果显示突变体mGIP243在昆明鼠体内具有显著的降血糖活性,当给药剂量为48nmol/kg时,其体内降血糖活性可延长至90分钟,相对于将第二位上的丙氨酸突变成丝氨酸的mGIP2和天然GIP半衰期显著延长。GIP类似物mGIP243在2型糖尿病模型鼠中也有降血糖活性,使其可能成为治疗糖尿病的候选药物。Glucose-dependent insulinotropic polypeptide（GIP）is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations.It＇s difficult to be applied because of its extremely short half life time.Modifying the dipeptidase IV recognition amino acid can prolong its half life time.Different GIP mutant genes that were obtained by artificial synthesizing or site-directed mutagenesis were cloned into the vector pET32a（＋）.These plasmids were expressed in Escherichia coli.The 6-histamine fused proteins were purified by NTA-Ni2＋ affinity chromatography,respectively.Purified fusion proteins were then digested with enterokinase and purified with another affinity chromatography and desalting.The biological activities of GIP analogs were tested in vivo in Kuming mice or diabetic db/db mice at a dose of 48nmol/kg by intraperitoneal injection associated with injection of glucose at 18mmol/kg.The results showed that the half life time of analog mGIP243 in Kunming mice was extended to 90 minutes（P0.001）,which was much longer than those of Ala to Ser analog mGIP2（30 minutes）or native GIP（shorter than 30 minutes）.The oral glucose tolerance tests in Kuming mice and db/db mice both supported the results with a magnificent glucose lowering effects（p0.01 VS saline group）by the end of a 60 minutes test.Interestingly,the novel GIP analog mGIP243 played good glucose lowering effect in diabetic mice.In conclusion,the novel GIP analog mGIP243 is a good drug candidate for diabetes.

关 键 词：葡萄糖依赖性促胰岛素释放肽(GIP) 抑胃肽 糖尿病 原核表达 血糖 

分 类 号：Q789[生物学—分子生物学]