日本血吸虫含EF-手型结构域钙结合蛋白的克隆表达与免疫诊断分析  

Cloning,Expression and Immunodiagnostic Analysis of Schistosoma japonicum Calcium-binding EF-hand Domain Containing Protein

作　　者：卢艳[1] 徐斌[1] 鞠川[1] 莫筱瑾[1] 陈绅波[1] 冯正[1] 王小宁[1] 胡薇[1] 徐斌 鞠川[2] 莫筱瑾[2] 陈绅波[2] 冯正[2] 王小宁[1,3] 胡薇[2,3] 

机构地区：[1]华东理工大学生物工程学院 [2]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025 [3]复旦大学生命科学学院,上海200433

出　　处：《中国寄生虫学与寄生虫病杂志》2012年第3期165-169,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家传染病科技重大专项(No.2009ZX1004-302);国家高技术研究发展计划(863计划)项目(No.2007AA02Z153);国际科技合作与交流专项(No.2010DFA33970)~~

摘　　要：目的克隆和表达日本血吸虫(Schistosoma japonicum)含EF-手型(EF-hand)结构域钙结合蛋白(SjEFCAB)的编码基因,纯化表达产物,鉴定重组蛋白的反应原性,初步评价其用于日本血吸虫病诊断的价值。方法以日本血吸虫虫卵cDNA文库中免疫筛选所获阳性克隆删除环化后的pBluescript-SjEFCAB质粒为模板,扩增SjEFCAB基因,将目的基因片段与原核表达载体pGEX-4T-1连接构建重组质粒pGEX-4T-1-SjEFCAB,转化至大肠埃希菌(Escherichia coli)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,用谷胱甘肽-S-转移酶(GST)标签亲和层析法纯化表达产物,蛋白质印迹(Western blotting)分析其反应原性。以SjEFCAB为包被抗原,采用间接ELISA方法检测日本血吸虫病(78份)、华支睾吸虫病(5份)、猪囊尾蚴病(10份)、卫氏并殖吸虫病(6份)和旋毛虫病(9份)患者血清,以及健康人血清(50份),评价该重组蛋白的免疫学诊断效果。结果构建了重组质粒pGEX-4T-1-SjEFCAB,并在E.coli BL21中高效表达,经亲和层析纯化获得可溶性重组蛋白SjEFCAB。Western blotting分析结果显示,重组蛋白SjEFCAB能被感染兔血清和血吸虫病患者血清所识别,在相对分子质量(Mr)约为8 200处出现条带。间接ELISA检测日本血吸虫病患者血清的敏感性为82.1%(64/78),特异性为95.0%(76/80)。与华支睾吸虫病患者、猪囊尾蚴病患者和旋毛虫病患者血清的交叉反应分别为1/5、1/10和1/9,与卫氏并殖吸虫病患者血清无交叉反应(0/6)。结论日本血吸虫SjEFCAB重组抗原具有较高的免疫学诊断价值。Objective To clone and expression Schistosoma japonicum calcium-binding EF-hand domain containing protein （SjEFCAB）, purify the expressed protein, and evaluate its antigenicity and diagnostic value. Methods The positive clone screened from egg cDNA library was used as template to amplify the SjEFCAB gene by PCR. The target fragment was cloned into prokaryotic expression vector pGEX-4T-1. The positive recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for expression of the protein. The recombinant protein was purified with GST-tag affinity chromatography. Western blotting was used to analyze the antigenicity. The purified protein was used as coating antigen for indirect ELISA to evaluate its diagnostic effect. Serum samples from patients with schistosomiasis japonica （78 cases）,clonorchiasis sinensis （5 cases）, cysticercosis （10 cases）, paragonimiasis westermani （6 cases）, trichinosis （9 cases） and healthy persons （50 cases） were examined. Results The recombinant plasmid pGEX-4T-1-SjEFCAB was constructed and the SjEFCAB recombinant protein （Mr 8 200） was expressed in E. coli. The soluble fusion protein was purified with affinity chromatography. Western blotting analysis showed that the recombinant protein was recognized by sera of in- fected rabbits and pooled sera of schistosomiasis japonica patients. The sensitivity and specificity of ELISA for diagnosis of schistosomiasis japonica were 82.1% （64/78） and 95.0% （76/80）, respectively. The cross reaction with sera of clonorchiasis sinensis, cysticercosis, and trichinosis patients were 1/5, 1/10, and 1/9, respectively. There was no cross reaction with sera of paragonimiasis westermani patients. Conclusion The recombinant SjEFCAB antigen has potential diagnostic value for schistosomiasis japonica.

关 键 词：日本血吸虫 EFCAB基因 免疫诊断 

分 类 号：R383.24[医药卫生—医学寄生虫学]