用EgAgB8/3重组蛋白ELISA-双抗夹心法建立棘球绦虫感染犬粪抗原检测系统的研究  

Development of Sandwich-ELISA detection system using recombinant protein of EgAB8/3 for diagnosis of dogs infected with Echinococcus spp

作　　者：陈洁[1,2] 马海梅[2] 古力帕丽.麦曼提依明 陈璐[1,2] 丁剑冰[2] 吾拉木.马木提 

机构地区：[1]新疆医科大学基础医学院寄生虫教研室,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院包虫病重点实验室,新疆乌鲁木齐830011

出　　处：《新疆医科大学学报》2011年第3期240-245,共6页Journal of Xinjiang Medical University

基　　金：国家自然科学基金项目(30760229);科技部"863计划项目"(2007AA02Z411)

摘　　要：目的高纯度表达细粒棘球绦虫抗原B(EgAgB8/3)重组蛋白,制备抗EgAgB8/3重组蛋白的多克隆抗体,建立ELISA-双抗夹心法检测棘球绦虫感染犬粪EgAgB8/3天然抗原系统。方法对已构建好的pET32a-EgAgB8/3-E.coli BL21(DE3)Lys S原核细胞表达系统进行IPTG诱导重组蛋白表达,用SDS-PAGE电泳分析鉴定重组蛋白的表达水平。目的蛋白用His-Binding-resin纯化柱进行纯化并免疫动物,免疫血清总IgG采用硫酸铵沉淀法纯化,一部分总IgG用过碘酸钠法进行标记,另一部分作为固相载体结合抗体备用。以未标记的总IgG为ELSA-双抗夹心法的包被抗体,细粒棘球绦虫感染犬粪粗提蛋白为夹心抗原,以标记的IgG为最终底物。结果成功获得高纯度EgAgB8/3重组蛋白和高效价多克隆抗体,运用标记和非标记IgG建立的ELSA-双抗夹心法对棘球绦虫感染犬粪特异性抗原检测的敏感性和特异性分别为85.0%和95.7%。结论获得的EgAgB8/3重组蛋白具有较好的免疫原性,且免疫动物后制备的多克隆抗体具有较高的敏感性和特异性。用EgAgB8/3重组蛋白EL-SA-双抗夹心法粪抗原检测系统的成功建立为棘球绦虫感染犬快速诊断试剂盒的制备提供了科学依据,此方法的稳定性和推广实用性将需用大量的流行病学调查资料进一步证实。Objective To express high purity EgAgB8/3 recombinant protein,to prepare anti recombinant EgAgB8/3 polyclonal antibodies and establishment of sandwich-ELISA test for detection of EgAgB8/3 native antigen in feces of dogs infected with Echinococcus spp.Methods A procaryotic protein expression system pET32a-EgAgB8/3-E.coli BL21（DE3） LysS was previously constructed in our laboratory was induced with isopropyl-1-thio-β-galactopyranoside（IPTG） for recombinant protein expression.The target protein was purified using His-Binding-resin column,and immunized to animals,serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method.A batch of purified IgGs were labeled using sodium iodide method,another batch of unlabeled IgGs were left as capture antibody.In Sandwich-ELISA system,the capture antibodies were used as coating antibody,the crude protein extract from dog feces infected with Echinococcus spp were used as sandwich antigens,and the labeled IgGs were used as final conjugate.Results The high purity EgAgB8/3 recombinant protein and the high titer mono-specific polyclonal antibodies were successfully prepared.The established Sandwich-ELISA system using labeled and unlabeled polyclonal antibodies revealed 85.0% sensitivity and 95.7% specificity to detect EgAgB8/3 native antigen in feces of dogs infected with Echinococcus spp.Conclusion The recombinant EgAgB8/3 protein has better immunogenic property,and the polyclonal antibodies obtained by animal immunization revealed high sensitivity and specificity.Successful development of the Sandwich-ELISA detection system using EgAgB8/3 recombinant protein is providing scientific data for preparation of fast diagnostic kit for dogs infected with Echinococcus spp.However,the stability and wide-range applicability of this method should be further evaluated using epidemiological mass data.

关 键 词：棘球绦虫 EgAgB8/3重组蛋白 多克隆抗体 ELISA-双抗夹心法 

分 类 号：R383.3[医药卫生—医学寄生虫学]