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目的 从日本血吸虫成虫cDNA文库中筛选并克隆日本血吸虫特异性IgE相关抗原编码基因。 方法 用ABC ELISA法从日本血吸虫病流行区筛选出高水平抗血吸虫成虫抗原IgE抗体的个体 15人 ,采集血清并混合。混合血清经Protein G柱吸收后 ,用于日本血吸虫成虫cDNA文库的免疫学筛选。PCR扩增阳性克隆插入cDNA片段。序列分析后 ,于该序列第一开读框两端设计引物并分别引入EcoRⅠ和NotⅠ位点 ,PCR扩增并纯化目的基因片段后克隆入质粒载体pGME T ,再亚克隆入表达载体pGEX 6p 1。经IPTG诱导表达 ,对表达产物进行SDS PAGE和Westernblotting鉴定。 结果 阳性克隆插入片段约 12 0 0bp ,第一开读框长 5 0 7bp ,编码 16 9个氨基酸 ,理论分子量为 19 3kDa。DNA序列分析显示 ,与已知序列同源性小于 4 0 %。重组质粒pGEX 6p 1/Sj4 3B能高效表达融合蛋白 ,且能被日本血吸虫特异性IgE抗体识别。 结论 成功构建的重组质粒pGEX 6p 1/Sj4 3B ,Sj4 3B可编码日本血吸虫特异性IgE抗体相关抗原
Objective To screen and clone Schistosoma japonicum specific IgE-associated antigen coding genes from adult Schistosoma japonicum cDNA library. Methods Fifteen individuals with high level of anti-IgE antibody to adult schistosomiasis were screened out from the endemic area of schistosomiasis japonica by ABC ELISA. Serum was collected and mixed. The mixed serum is absorbed by Protein G column for immunological screening of adult Schistosoma japonicum cDNA library. PCR amplification of positive clones insert cDNA fragments. After sequence analysis, primers were designed at both ends of the first open reading frame of the sequence and introduced into EcoRI and Notl sites respectively. The target gene fragment was amplified by PCR and cloned into the plasmid pGME T and subcloned into the expression vector pGEX 6p 1 . After induced by IPTG, the expressed products were identified by SDS PAGE and Western blotting. Results The positive cloned insert was about 1200 bp, the first open reading frame was 577 bp, encoding 16 9 amino acids with a theoretical molecular weight of 19 3 kDa. DNA sequence analysis showed less than 40% homology with known sequences. The recombinant plasmid pGEX 6p 1 / Sj4 3B could express the fusion protein efficiently and could be recognized by Schistosoma japonicum specific IgE antibody. Conclusion The constructed recombinant plasmids pGEX 6p 1 / Sj4 3B and Sj4 3B can encode the specific antigen of Schistosoma japonicum specific IgE antibody