免疫印迹法测定Tp47蛋白与梅毒患者血清的免疫反应性  

Immunoreactivity analysis of Tp47 recombinant protein of Treponema Pallidum by Western-Blot assay

作　　者：郑和平[1] 覃晓琳[1] 黄进梅[1] 薛耀华[1] 白顺[1] 吕萍[1] 杨斌[1] 

机构地区：[1]广东省皮肤性病防治中心,广东广州510091

出　　处：《国际检验医学杂志》2015年第10期1327-1329,共3页International Journal of Laboratory Medicine

基　　金：广东省科技计划项目(2011B031800225;2012B031800175)

摘　　要：目的克隆Tp47基因、构建原核表达载体、表达并纯化Tp47蛋白,通过免疫印迹法(Western-Blot)检测其免疫反应活性。方法聚合酶链反应扩增Tp47基因,将Tp47克隆至pGEX-6P-1载体,构建重组表达质粒pGEX-6P1-Tp47,经测序鉴定正确后,转化大肠埃希菌BL21(DE3),经诱导表达后,用亲和层析方法纯化重组蛋白,鉴定正确后采用Western-Blot检测重组蛋白与梅毒阴性、阳性血清的免疫反应性。结果成功构建pGEX-6P1-Tp47原核表达质粒,表达、纯化后获得了相对分子质量约为71×103的重组蛋白;Western-Blot检测显示其能与梅毒阳性患者血清发生特异性反应,而与梅毒阴性患者血清无交叉反应性。结论成功克隆、表达、纯化Tp47重组蛋白,其与临床各期梅毒患者血清具有较好的免疫反应性,为Tp47重组蛋白用于梅毒早期诊断提供了理论依据。Objective To clone ,construction ,express and purify Tp47 of Treponema pallidum （Tp） ,and assess the immunoreac‐tivity by Western‐Blot .Methods Tp47 was amplified by polymerase chain reaction ,and then cloned to the vector pGEX‐6P‐1 .The correct sequence of the recombinant plasmids pGEX‐6P1‐Tp47 was transformed into Escherichia coli BL21 （DE3） and induced .The expression product was analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis and Western‐Blot .The expression protein was purified .Serum of different clinical stages of syphilis was used as the antibody to detect the immunoreactivity of the protein by Western‐Blot .Results A fusion protein with molecular weight about 71 ＆#215; 103 was attained .Western‐Blot proved that the recombinant protein can react with Tp IgG positive sera .And the specificities and sensitivities of the diagnostic reagent detected by sera were 100% .Conclusion The recombinant protein Tp47 was expressed and purified with good antigen activity ,which could provide the basis of theory and practice for the development of early diagnostic kit applying to detect Tp infection .

关 键 词：梅毒 重组蛋白 免疫印迹法 

分 类 号：R759.1[医药卫生—皮肤病学与性病学] R446.6[医药卫生—临床医学]