日本血吸虫特异性IgE相关抗原编码基因的克隆和鉴定  

Clonning and Identification of the Gene Encoding Specific IgE Antibody\|Related Antigen of Schistosoma japonicum

作　　者：王勇[1] 苏川[1] 张兆松[1] 胡雪梅[1] 沈蕾[1] 刘丰[1] 王荣芝[1] 陈淑贞[1] 李春林[1] 吴观陵[1] 

机构地区：[1]南京医科大学医学分子生物学研究所分子免疫寄生虫学研究室,南京210029

出　　处：《中国寄生虫学与寄生虫病杂志》2001年第5期272-275,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省重点实验室开放课题经费资助 (No.80 2 3 )

摘　　要：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 ,Sj4Objective To obtain the gene encoding specific IgE\|related antigen of Schistosoma japonicum from the cDNA library of adult Schistosoma japonicum . Methods The pooled sera from 15 individuals with high levels of specific IgE antibody against SWAP were absorbed with Protein\|G and used for screening of IgE\|related antigen from the adult worm cDNA library of Schistosoma japonicum . The inserted cDNA was amplified by PCR and sequenced. According to the first reading frame of the sequence, a pair of new probes, in which Eco R Ⅰ and Not Ⅰsites were incorporated respectively,were designed and used to amplify the target gene. Then,the gene was cloned into vector pGEM\|T and subcloned into expression vector pGEX\|6p\|1. The fusion protein was expressed, analysed by SDS\|PAGE and identified by Western blotting with the specific IgE antibody, respectively. Results The inserted cDNA fragment from the positive clone was about 1 200 bp,with the ORF of 507 bp which encoded 169 amino acids. The deduced molecular weight of the recombinant protein was 19\^3 kDa. The homology between the target gene(Sj43B) and other known DNA sequences was less than 40%. The fusion protein expressed by the recombinant vector pGEX\|6p\|1/Sj43B could be recognized by schistosome specific IgE antibody. Conclusion Sj43B may encode the specific IgE\|related antigen of Schistosoma japonicum . The successful construction of recombinant plasmid pGEX\|6p\|1/Sj43B lay the groundwork for further studies on immunological characteristics and protection immunity of the recombinant protein.

关 键 词：日本血吸虫 IGE 基因克隆 重组抗原 融合蛋白 鉴定 

分 类 号：R383.24[医药卫生—医学寄生虫学]