摘要
目的 :构建、表达、纯化日本血吸虫铁蛋白基因的原核表达重组蛋白 ,并初步观察其免疫诊断价值。方法 :将日本血吸虫铁蛋白基因亚克隆于pGMC载体 ,转化重组体到大肠杆菌E .coli 2 5 6 6进行表达 ;采用电泳层析法分离纯化日本血吸虫铁蛋白 ;用SDS PAGE和Westernblot鉴定表达纯化产物 ;用Dot ELISA分别检测了血吸虫病人及正常人的血清。结果 :重组的日本血吸虫铁蛋白以GMC(粒细胞巨噬细胞集落刺激因子 ) 日本血吸虫铁蛋白融合蛋白的形式在细菌中得到高效表达 ,分子量为 4 0kD ,能被日本血吸虫感染兔血清识别。用重组铁蛋白检测 30例日本血吸虫病人血清和 30例正常人血清 ,阳性率和假阳性率分别为 93 3%和 3 3%。结论 :成功地表达纯化日本血吸虫重组铁蛋白 ,且该蛋白具有一定的免疫诊断价值。
Objective:To construct,express and purify the prokaryotic expressed recombinant protein which contains Schistosoma japonicum ferritin gene,and to explore its use in immunodiagnosis.Methods:The ferritin gene was subcloned into the expression vector pGMC.The expression vector pGMC with ferritin gene(pGMC-Sjferritin) was then used to transfer E.coli strain ER2566.Fractionate and purify pGMC-Sjferritin using electrophoretic chromatography.Molecular weight of pGMC-Sjferritin was determined by SDS-PAGE and Western blot.The protein used an antigen for Dot-ELISA to detect sera from schistosomiasis patients and normal controls.Results:The recombinant protein pGMC-Sjferritin was expressed high efficiently in E.coli ER2566 and its molecular weight was 40 kD.The protein could be recognized by antibody in sera of rabbit infected with Schistosoma japonicum .The sera from 30 schistosomiasis patients and 30 normal controls were tested.The result showed the positive rate and the false-positive rate were 93 3% and 3 3%,respectively.Conclusion:The authors have successfully expressed and purified the recombinant protein which contains Schistosoma japonicum ferritin gene.The recombinant protein can be used an antigen for the immunodiagnosis of schistosomiasis.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2004年第10期696-699,共4页
Chinese Journal of Immunology
基金
WHO/TDR资助项目(No .980 2 5 5 )
湖南省科委基金资助项目 (No .0 0jzy 2 115 )
关键词
日本血吸虫
铁蛋白
基因克隆
表达
免疫诊断
Shistosoma japonicum
Ferritin
Gene cloning
Expression
Immunodiagnosis
