人羧基肽酶H原核表达载体的构建及其应用  

Construction of prokaryotic expression vector for human carboxypeptidase H and its application

作　　者：向斌[1] 周智广[1] 黄干[1] 杨琳[1] 张翼[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所中南大学糖尿病中心,长沙410011

出　　处：《中国糖尿病杂志》2007年第2期84-86,共3页Chinese Journal of Diabetes

基　　金：国家自然科学基金资助项目(30400217);湖南省科技厅重点项目(03SSY1009)

摘　　要：目的构建人羧基肽酶H(CPH)的原核表达系统,以获得其重组蛋白,并初步应用于人CPH自身抗体(CPH-Ab)的检测。方法采用PCR方法,从质粒pcDNAⅡ扩增出CPH基因片段,将其克隆到原核表达载体pQE30后转化宿主菌E.coliM15,IPTG诱导CPH蛋白表达,用Ni-NTA亲和层析柱纯化,Western-blot法检测表达蛋白的抗原性。用重组CPH蛋白作为包被抗原,初步建立检测CPH-Ab的ELISA方法。结果获得了CPH原核表达载体,CPH蛋白得到了表达。纯化的蛋白可被CPH-Ab阳性的1型糖尿病患者血清识别。与放射配体法相比较,以重组CPH蛋白作为包被抗原初步建立的ELISA方法,其灵敏性和特异性均升高,分别为93.3%和97.5%。结论重组CPH蛋白具有良好的抗原性,为糖尿病患者CPH-Ab的检测奠定了基础。Objective To get human recombinant carboxypeptidase H （CPH） protein,and to explore the application of the recombinant protein. Methods The human CPH gene fragment was amplified from plasmid pcDNA Ⅱ by PCR and cloned into prokaryotic expression vector pQE30 . The recombinant expression plasmid was transfeeted into E. coli M15 and induced with IPTG. CPH protein was purified by Ni-NTA column and the antigenieity of the purified protein was confirmed by Westernblot. The purified recombinant protein was used in ELISA to evaluate the method for the detection of CPH-Ab in human sera. Results Prokaryotic expression vector for CPH was successfully constructed. The purified protein could be recognized by the CPH Ab in sera of type 1 diabetic patients. When compared with radioligand assay, the more increased sensitivity and specificity of the ELISA test were 93.3% and 97.5 %, respectively. Conclusions The purified CPH protein is one of suitable candidates of immunodiagnostic antigen.

关 键 词：糖尿病 自身免疫性 羧基肽酶H 重组蛋白质类 

分 类 号：R587.1[医药卫生—内分泌]