垂体腺苷酸环化酶激活多肽38及其衍生物的表达、纯化和活性  

Expression,Purification and Activity of Pituitary Adenylate Cyclase-activting Polypeptide 38 and Its Derivative

作　　者：张洪丹[1] 肖磊[1] 赵丽芬[1] 王小溪[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

出　　处：《中国生物制品学杂志》2011年第5期526-530,共5页Chinese Journal of Biologicals

摘　　要：目的提高垂体腺苷酸环化酶激活多肽38(Pituitary adenylate cyclase-activating polypeptide 38,PACAP38)的体内稳定性、延长其生物学活性。方法对PACAP38的氨基酸序列进行改造(命名为PACAP38衍生物cPACAP38),并根据大肠杆菌密码偏爱性设计并合成PACAP38及其衍生物的基因片段;分别将两基因片段克隆至表达载体pET-32a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达;表达的融合蛋白经镍离子亲和层析纯化,肠激酶切割获得目的多肽;通过小鼠饮食抑制试验比较两多肽的体内活性;长期给药观察小鼠腹腔糖耐量及小鼠血清甘油三酯、HDL、LDL和胆固醇的变化。结果两种融合蛋白Trx-PACAP38和Trx-cPACAP38的表达量分别占菌体总蛋白的22%和25%,为可溶性表达,纯化后纯度分别为90%和98%;PACAP38及cPACAP38均有抑制小鼠饮食的作用,cPACAP-38的效果优于PACAP38;连续给药10 d后,小鼠腹腔糖耐量、血清甘油三酯、HDL、LDL、胆固醇均无明显变化。结论已成功获得体内活性更稳定的PACAP38衍生物,为PACAP38的基础研究及应用奠定了基础。Objective To improve the in vivo stability and prolong the half-life of bioactivity of pituitary adenylate cyclase-activting polypeptide 38（PACAP38）.Methods The amino acid sequence of PACAP38 was modified to obtain a derivative named cPACAP38.The gene fragments encoding PACAP38 and cPACAP38 were designed and synthesized according to the codon bias of E.coil and cloned into vector pET-32a（＋）.The constructed recombinant plasmids were transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed fusion proteins were purified by nickel ion affinity chromatography and digested with enterokinase,by which two target polypeptides were obtained and tested for anorexigenic activities in mice.Mice were treated with the polypeptides for 10 d,then determined for intraperitoneal glucose-resistance as well as serum triglyceride,HDL,LDL and cholesterol contents.Results Fusion proteins Trx-PACAP38 and Trx-cPACAP38 were expressed in soluble forms,which contained 22% and 25% of total somatic proteins and reached purities of 90% and 98% after purification respectively.Both PACAP38 and cPACAP38 showed anorexigenic activities in mice.However,the anorexigenic activity of cPACAP38 was higher than that of PACAP38.No significant changes were observed in intraperitoneal glucose-resistance as well as serum triglyceride,HDL,LDL and cholesterol contents of mice 10 d after treatment with the fusion proteins.Conclusion The derivative of PACAP38 with higher in vivo activity was successfully expressed,which laid a foundation of basic study and application of PACAP38.

关 键 词：垂体腺苷酸环化酶激活多肽 饮食抑制 长期给药 葡糖耐量试验 

分 类 号：Q555[生物学—生物化学] R392-33[医药卫生—免疫学]