日本血吸虫尾蚴cDNA文库的免疫学筛选及EST序列的分析  

Immunological Screening of Schistosoma japonicum Cercariae cDNA Library and the EST Strategy

作　　者：陈奕慧[1] 李孜[1] 李涛[1] 李巧燕[1] 麦璟莹[1] 

机构地区：[1]广州医学院病原生物学教研室,广州510182

出　　处：《热带医学杂志》2007年第1期46-48,54,共4页Journal of Tropical Medicine

基　　金：广东省自然科学基金(No.5300973)。

摘　　要：目的为获得日本血吸虫(Schistosoma japonicum,Sj)转化生长因子β1(TGF-β1)基因的部分或全长cDNA序列,同时验证用针对某一蛋白的抗体筛选表达文库是否可以得到相应蛋白对应的基因序列。方法采用兔抗鼠TGF-β1血清,对Sj尾蚴cDNA表达文库进行免疫学筛选,对阳性克隆进行PCR扩增后,将大于500bp的克隆进行复筛,对复筛阳性的克隆进行测序和生物信息学鉴定。结果对大约106个噬菌斑进行了初筛,共获得7个阳性克隆;经过PCR扩增后,其中有4个克隆大于500bp,复筛获得3个持续阳性克隆;对测序后的阳性克隆进行生物信息学鉴定,得到的三个克隆均与日本血吸虫辅酶Q10氧化还原酶(SjCHGC)基因具有高的同源性(Blastn分值都大于200)。结论SjCHGC蛋白与兔抗鼠TGF-β1抗体的反应为交叉反应,用抗某一蛋白的抗体筛选表达文库得到相应蛋白的基因序列的方法不一定可行。Objective To determine the sequence of transforming growth factor β（TGF-β） gene from Schistosoma japonicum （Sj）, and to determine whether the antibody specific for the murine TGF-β can be used in the screening of cDNA expression library for the Sj TGF-β. Methods The cDNA library of Sj cercariae was screened with rabbit anti- mouse TGF-β1 antibody, and the inserts from the positive clones were specifically amplified by PCR. Gene segments that exceeded 500 bp were re-screened. DNA form the positive clones was sequenced and then compared with the database from the GenBank. Results Of the 10^6 immtmo-screened phage plaques, 7 positive cDNA clones were obtained. Four of these clones, with a size of greater than 500 bp, were successfully amplified by PCR. Three stable positive clones showed homology with the Sj NADH-ubiquinone oxidoreductase （CHGC） （Blasto score 〉200）. Conclusion Cross- reaction between the anti-mouse TGF-β1 antibody and Sj CHGC protein was observed. The antibody specific for TGF- β1 probably can not be used to obtain the corresponding gcne sequence from the Sj cDNA expression library.

关 键 词：日本血吸虫 免疫学筛选 TGF-Β 

分 类 号：R383.24[医药卫生—医学寄生虫学]