日本血吸虫毛蚴侵袭前后湖北钉螺差异表达cDNA文库的构建与分析  

Construction and analysis of differentially expressed cDNA library for Oncomelania hupensis pre-and post-exposed to miracidium of Schistosoma japonicum

作　　者：刘琴[1] 李石柱[1] 张仪[1] 周晓农[1] 

机构地区：[1]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,上海200025

出　　处：《中国血吸虫病防治杂志》2010年第6期539-543,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家重大科技专项(2008ZX10004-011);上海市自然科学基金(10ZR1433400);中国博士后科学基金(20070420422)

摘　　要：目的构建湖北钉螺被日本血吸虫毛蚴侵袭前、后差异表达cDNA文库,筛选湖北钉螺免疫相关分子,为探讨病原与中间宿主的相互作用奠定基础。方法提取被日本血吸虫毛蚴侵袭前、后湖北钉螺的头足部组织总RNA,纯化mR-NA,反转录合成cDNA。分别以被日本血吸虫毛蚴侵袭前湖北钉螺(未处理组)和被日本血吸虫毛蚴侵袭后湖北钉螺(处理组)的头足部组织作为检测方(Tester)和驱动方(Driver),利用PCR方法选择cDNA杂交试剂盒分别进行正向和反向抑制性消减杂交。将获得的正向抑制性消减杂交产物克隆入pGEM-T载体,重组质粒转入E.coliDH5α,对菌液用PCR法扩增鉴定其中的插入片段。随机抽取354个阳性克隆进行DNA序列分析,将所得表达序列标签(ESTs)序列在线进行BLAST分析。结果从正向文库随机挑取的354个阳性克隆中测得350个ESTs序列,生物信息学分析发现了34个湖北钉螺新基因,其中1个与已知基因部分同源。结论成功建立了被日本血吸虫侵袭前、后湖北钉螺差异表达片段cDNA消减文库,发现了湖北钉螺新基因,为筛选与湖北钉螺天然免疫相关的分子、进一步探讨中间宿主与病原的相互作用及筛选血吸虫病传播阻断疫苗候选分子奠定了基础。Objective To construct a differentially expressed cDNA library of Oncomelania hupensis,the intermediate host of Schistosoma japonicum,for pre-and post-exposed to miracidium of S.japonicum,and sequentially to screen the immunity-related molecules and lay the foundation for studying the interaction between pathogen and intermediate hosts.Methods The total RNA samples were separately isolated from pools of head-foot tissues of O.hupensis which were respectively pre-and post-exposed to miracidium of S.japonicum,then mRNA were purified and the cDNA were reverse transcription.The head-foot tissues of O.hupensis which were pre-and post-exposed to miracidium of S.japonicum were acted as driver and tester,respectively.And suppression subtractive hybridization（SSH）was performed by using the PCR-Select cDNA Subtraction kit.The forward subtracted cDNA was ligated into pGEM-T easy vector.Recombinant plasmids were transformed into competent Escherichia coli cells DH5α and the inserted fragments were identified by Bacterium PCR.Three hundred and fifty-four positive clones randomly selected were for DNA sequence analysis,and the obtained ESTs were analyzed using online BLAST sequence analysis.Results A total of 354 randomly selected positive clones from the forward library were sequenced and 350 valid ESTs were obtained.Thirty-four O.hupensis new genes were discovered by bioinformatics analysis,of which one was homologous with known genes.Conclusions The subtractive cDNA library of differentially expressed fragments of O.hupensis for pre-and post-exposed to miracidium of S.japonicum are constructed,and new genes of O.hupensis are discovered.This will lay the foundation for screening O.hupensis immune molecules related to the natural elements and further studies on the interaction between host and pathogen and screening of transmission-interrupted vaccine candidate.

关 键 词：湖北钉螺 日本血吸虫 抑制消减杂交 差异表达cDNA文库 

分 类 号：R383.24[医药卫生—医学寄生虫学]