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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 cDNA library of Coix japonicus infected with mirabilis japonicum before and after its invasion, and to screen for the immune-related molecules of Oncomelania hupensis in Hubei Province, and to lay the foundation for exploring the interaction between the pathogen and the intermediate host. Methods The total RNA was extracted from the head and foot tissues of snails of Schistosoma japonicum before and after the invasion of Schistosoma japonicum. The mR-NA was purified and cDNA was reverse transcribed. The head and foot tissues of Hubei snail (untreated group) and Schistosoma japonicum (treated group) were used as Tester and Driver respectively before the invasion of Schistosoma japonicum by miracidia, and PCR was used to select cDNA hybridization kit were forward and reverse inhibitory subtractive hybridization. The obtained positive inhibitory subtractive hybridization product was cloned into pGEM-T vector, and the recombinant plasmid was transformed into E. coli DH5α, and the inserted fragment was identified by PCR amplification. A total of 354 positive clones were randomly selected for DNA sequence analysis. The resulting ESTs sequences were subjected to BLAST analysis online. Results A total of 350 ESTs were sequenced from 354 positive clones randomly collected from the forward library. Bioinformatics analysis revealed 34 new snails from Hubei and one of them was partially homologous with the known gene. Conclusion The cDNA subtractive library of Snail from Hubei Province was established successfully before and after the invasion of Schistosoma japonicum, and the new gene of Snail from Hubei Province was found. In order to screen the molecules related to innate immunity of Oncomelania hupensis, further explore the interaction between the host and the pathogen and screen the Schistosoma japonicum Disease transmission blocks vaccine candidate molecules laid the foundation.