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目的克隆并表达日本血吸虫肌醇单磷酸酶(SjIM)编码基因cDNA,评估该重组抗原抗血吸虫感染的免疫保护效果。方法以日本血吸虫42 d虫体cDNA为模板,经PCR扩增编码SjIM蛋白的开放阅读框(ORF)基因片段。采用荧光实时定量PCR分析该基因在童虫和成虫的表达情况。以pET28a(+)为载体构建重组表达质粒,经异丙基βD硫代吡喃半乳糖苷诱导,制备重组SjIM蛋白。Western blotting检测重组蛋白的抗原性与免疫原性,利用重组SjIM抗原免疫小鼠评估其免疫保护效果。结果获得了编码SjIM基因ORF的cDNA片段,ORF长度为834 bp,编码278个氨基酸。荧光实时定量PCR显示该基因在35 d虫体中表达量最高。获得了SjIM重组蛋白,其具有良好的免疫原性。免疫组小鼠获得48.76%的减虫率和41.29%的肝脏减卵率。结论获得了日本血吸虫SjIM基因ORF的cDNA序列,并制备了重组SjIM蛋白,该重组抗原在小鼠体内能诱导产生部分抗血吸虫感染的免疫保护效果。
Objective To clone and express SjIM cDNA of Schistosoma japonicum, and evaluate the immunoprotective effect of the recombinant antigen against Schistosoma japonicum infection. Methods The open reading frame (ORF) gene encoding SjIM protein was amplified by PCR using the 42 d worm cDNA of Schistosoma japonicum as a template. Fluorescence real-time quantitative PCR was used to analyze the expression of the gene in Schistosoma japonicum and adult worms. Recombinant plasmids were constructed by using pET28a (+) as a vector and induced by isopropyl βD thiogalactopyranoside to prepare recombinant SjIM protein. Western blotting was used to detect the antigenicity and immunogenicity of the recombinant protein. Immunization of mice with recombinant SjIM antigen was used to evaluate the immunoprotection. Results The cDNA fragment encoding the ORF of SjIM gene was obtained. The length of ORF was 834 bp, encoding 278 amino acids. Real-time fluorescence quantitative PCR showed that the gene was expressed the highest in 35 d worms. SjIM recombinant protein was obtained which has good immunogenicity. The immunized mice achieved a worm reduction rate of 48.76% and a liver ejaculation rate of 41.29%. Conclusions The cDNA sequence of SjIM gene of Schistosoma japonicum was obtained and a recombinant SjIM protein was prepared. The recombinant antigen could induce the partial immunosuppressive effect of anti-schistosome infection in mice.