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目的克隆、表达日本血吸虫Toll样受体相互作用蛋白(SjTollip)基因,并研究该基因在日本血吸虫各虫期的转录表达情况。方法从日本血吸虫cDNA文库中挑出Toll样受体相互作用蛋白基因进行体外扩增,并克隆入原核表达载体pET-28a,转化大肠埃希菌BL21(DE3),异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,用组氨酸标签亲和层析法纯化表达产物。制备日本血吸虫各虫期阶段总RNA和重组SjTollip蛋白免疫新西兰白兔的免疫兔血清,利用RT-PCR和蛋白质免疫印迹分析SjTollip基因在日本血吸虫各期转录表达的差异及重组SjTollip蛋白的免疫原性。结果构建了重组原核表达载体SjTollip/pET-28a,诱导获得以包涵体形式表达的不可溶重组蛋白,相对分子质量约为24000,与预测的融合蛋白相对分子质量相符。纯化后的重组蛋白rSjTollip可被日本血吸虫感染兔血清识别。虫期转录特异性分析显示,该基因在尾蚴和雄虫中转录水平较低。免疫印迹分析结果显示,在虫卵、尾蚴、童虫、雄虫、雌虫各发育阶段都检测到该蛋白,但童虫和雌虫中表达水平明显升高。结论 SjTollip基因在日本血吸虫各发育阶段转录表达水平有较大差异,其有可能成为潜在的日本血吸虫病疫苗候选抗原和药物及诊断靶点。
Objective To clone and express the Toll-like receptor interacting protein (SjTollip) gene of Schistosoma japonicum and study the transcriptional expression of this gene at each stage of Schistosoma japonicum. Methods The Toll-like receptor interacting protein gene was amplified from the cDNA library of Schistosoma japonicum and amplified by in vitro cloning into the prokaryotic expression vector pET-28a. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), isopropyl-β-D Inducible expression of galactoside (IPTG) was detected by histidine tag affinity chromatography. The total RNA of each stage of Schistosoma japonicum and the immunized rabbit serum of recombinant SjTollip protein immunized New Zealand white rabbits were prepared. The differences of transcriptional expression of SjTollip gene in various stages of Schistosoma japonicum and the immunogenicity of recombinant SjTollip protein were analyzed by RT-PCR and Western blotting . Results The recombinant prokaryotic expression vector SjTollip / pET-28a was constructed and the insoluble recombinant protein was expressed in inclusion bodies. The relative molecular mass was about 24000, which was in good agreement with the predicted molecular weight of the fusion protein. The purified recombinant protein rSjTollip can be recognized by Schistosoma japonicum infected rabbit serum. Transcriptional specificity analysis of the worm stage showed that the gene was lower in cercariae and males. Western blot analysis showed that the protein was detected in all stages of eggs, cercariae, schistosomula, male and female, but the expression levels were significantly increased in schistosomiasis and females. Conclusion The transcriptional level of SjTollip gene in different developmental stages of Schistosoma japonicum is quite different, which may become a potential candidate antigens and drugs and diagnostic targets for schistosoma japonicum vaccine.