多房棘球绦虫ELP抗原在大肠埃希菌中的表达及其在免疫诊断中的初步应用  

Expression of ELP protein of Echinococcus multilocularis in Escherichia coli and its use in immunodiagnostic assay

作　　者：王昌源[1] 张洪花[2] 陈雅棠[3] 李文桂[3] 唐霓[3] 

机构地区：[1]山东大学医学院传染病学教研室,济南市传染病院,山东济南250021 [2]青岛市疾病预防控制中心 [3]重庆医科大学

出　　处：《中国病原生物学杂志》2006年第2期98-101,共4页Journal of Pathogen Biology

基　　金：重庆市卫生局重点项目(No.99-1003);山东大学博士基金联合资助(2003)。

摘　　要：目的实现多房棘球绦虫ELP蛋白在大肠埃希菌中的表达,制备高纯度、高特异性的重组抗原,为多房棘球绦虫感染的流行病学调查和临床诊断提供新的工具。方法在建立了多房棘球绦虫elp基因克隆的基础上,应用亚克隆方法将目的基因插入原核非融合表达载体pQE30(+)。经酶切鉴定,转化于大肠埃希菌,以IPTG进行诱导表达。SDS-PAGE及Westernblot进行鉴定。亲和层析纯化ELP蛋白,用于ELISA检测患者血清特异性抗体。结果酶切鉴定、SDS-PAGE及Westernblot分析证实,成功地构建了pQ-ELP原核非融合表达载体,该载体转化大肠埃希菌在IPTG诱导下能高效表达ELP蛋白。ELP重组抗原用于ELISA检测18份包虫病人血清均阳性,10份华支睾吸虫病、27份乙肝病毒感染者和10份健康人血清均为阴性。结论多房棘球绦虫ELP抗原在大肠埃希菌中得到了高效非融合表达,初步实验结果显示该重组蛋白对包虫病具有较高的诊断价值。Objective To realize the expression of Echinococcus multilocularis ELP protein in Escherichia coli, so as to obtain high purity and specificity recombinant antigen, and provide a new tool for epidemiological survey and clinical diagnosis of E. rnultilocularis infection. Methods Based on the successful cloning of elp gene of E. multilocularis and subcloning technique, the target elp gene was inserted into a prokaryotic non fusion expression vector pQE30（＋）. The recombinant plasmid was analyzed with restriction-endonuclease digestion and then transformed into host E. coli, SG13009. The expressed recombinant ELP induced with isopropylthio-β-D-galactoside （IPTG）was examined by SDS-PAGE and Western blot. ELP protein was purified with affinity chromatography and used in ELISA to detect specific antibody in patients＇ sera. Results Restriction endonuclease analysis, SDS-PAGE and Western blot proved that a construct expressing non-fused ELP protein was successfully established. When the recombinant plasmid was transformed into E. coli and induced with IPTG, it could efficiently express ELP protein. In ELISA with the recombinant antigen, 18 sera from patient with hydatid disease were all positive, and negative results were obtained with sera from 10 cases of clonorchiasis sinensis, 27 cases of hepatitis B and 10 healthy people. Conclusion Non-fusion form of E. multilocularis ELP protein has been highly expressed in E. coli. A preliminary experimental result indicates the recombinant protein ELP has a higher diagnostic value for E. rnultilocularis infection.

关 键 词：多房棘球绦虫 ELP抗原 非融合表达 免疫诊断 

分 类 号：R383.33[医药卫生—医学寄生虫学]