日本血吸虫Toll样受体相互作用蛋白 基因的克隆 表达及鉴定

Cloning,expression and identification of Toll like receptor interacting protein gene of Schistosoma japonicum

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 Schistosoma japonicum Toll like receptor interacting protein （SjTollip） in prokaryotic expression system and analyze its stage-specific transcription and expression. Methods The encoding sequence selected from S. japonicum cDNA library was amplified by PCR. The SjToUip gene obtained was subcloned into pET-28a, then transformed into E.coli BL21 and induced with IPTG for expression. The expressed protein was purified with Ni-NTA resin. Total RNA were extracted from different stages of S. japonicum. The immune rabbit sera were prepared by immunizing New Zealand white rabbits with purified recombinant SjTollip protein. RT-PCR and Western blotting were used to analyze the transcription and expression level at the different stages and the immunogenicity. Results The expression vector of SjTollip/pET-28a was constructed and expressed as inclusion bodies （Mr 24 000）. The recombinant protein rSjTollip was specifically recognized by the S. japonicum-infected rabbit serum. SjTollip showed lower transcription level in stages including cercariae and male worms. Western blotting analysis showed that the target protein was detected in all stages. The expression level in stages including schistosomulum and female worm was much higher. Conclusions The SjTollip transcription and expression level at the different stages of S. japonicum is different, and this gene might be a potential candidate for target of vaccine, drug and diagnosis.