日本血吸虫Tollip蛋白和SSX蛋白作为诊断和疫苗候选抗原的研究

发布时间：2018-11-08 09:24

【摘要】：血吸虫病是免疫性疾病,血吸虫性别特异分化、生殖产卵等阶段对其生存传播有重要影响。本课题组前期筛选出几十个可能与日本血吸虫免疫、性别及生殖相关的蛋白,作为日本血吸虫诊断抗原与疫苗的候选靶标。本研究针对其中的SjTollip(AY814053, AAW25785.1)和SjSSX(BU793503, CAX76185.1)2个蛋白进行研究。主要研究内容包括：(1)根据序列信息进行以下生物信息学分析：预测信号肽、预测跨膜区域、预测功能位点和结构域、同源序列搜索、多序列比对以及构建进化树等；(2)基因的克隆、原核表达以及重组蛋白的纯化；(3)重组蛋白免疫原性检测,制备重组蛋白兔多克隆抗体并评价,考察基因的虫期转录特异性和蛋白的虫期表达特异性；(4)蛋白质的诊断价值评价和动物保护性实验。 Tollip是TLRs信号通路中的负性调节因子,它能够抑制过度和长时间的免疫反应。SjTollip为相对保守蛋白,在多个物种中有同源序列。构建了pET-28a/SjTollip重组质粒,诱导表达获得不可溶蛋白,通过尿素纯化获得较高浓度和纯度的重组蛋白；Western blot检测显示SjTollip具有一定的免疫原性；SjTollip免疫兔多克隆抗体的滴度达到1：256,000；SjTollip在毛蚴、胞蚴、虫卵、童虫、雌虫中转录水平高,尾蚴和雄虫中转录水平低；SjTollip主要在童虫和雌虫中表达,与基因转录一致；rSjTollip作为包被抗原通过ELISA方法检测不同人群血清,敏感性和特异性分别为70%和80%；用rSjTollip免疫小鼠诱导产生58.05%的减虫率,感染后40d-42d粪便减卵率为59.84%,43d～45d粪便减卵率为78.68%。说明其能够诱导产生较强的免疫保护力,是潜在的疫苗候选靶标。 SSX蛋白是CT抗原家族成员,CT抗原的异位表达可以导致自体统一细胞或体液产生免疫应答,可用于构建和发展免疫治疗方案,作为免疫治疗的靶点。SjSSX与日本血吸虫重要宿主中的同源序列的相似性较低,构建了pET-28a/SjSSX重组质粒,诱导表达获得不可溶蛋白,通过尿素纯化获得较高浓度和纯度的重组蛋白；Western blot检测显示SjSSX具有良好的免疫原性；SjSSX免疫兔多克隆抗体的滴度达到1：256,000;SjSSX在童虫、雌虫中转录水平高,胞蚴、虫卵次之,毛蚴、尾蚴和雄虫中转录水平低；SjSSX在虫卵、尾蚴、童虫、雄虫、雌虫均有表达；SjSSX作为包被抗原通过ELISA方法检测不同人群血清,敏感度为80%,特异度为90%；用SjSSX免疫小鼠可诱导产生28.16%的减虫率,感染后43d-45d粪便减卵率为4.25%。
[Abstract]:Schistosomiasis is an immune disease. Sex specific differentiation of schistosomiasis and reproductive oviposition play an important role in the survival and transmission of schistosomiasis. Dozens of proteins that may be related to Schistosoma japonicum immunity, sex and reproduction were screened by our research group, which were used as candidate targets for diagnostic antigen and vaccine of Schistosoma japonicum. In this study, two proteins, SjTollip (AY814053, AAW25785.1) and SjSSX (BU793503, CAX76185.1), were studied. The main contents are as follows: (1) the following bioinformatics analysis is carried out according to the sequence information: predicting signal peptide, predicting transmembrane region, predicting functional site and domain, homologous sequence searching, multi-sequence alignment and constructing evolutionary tree; (2) cloning of gene, prokaryotic expression and purification of recombinant protein; (3) immunogenicity of recombinant protein, preparation and evaluation of rabbit polyclonal antibody against recombinant protein; (4) the diagnostic value of protein and animal protective experiment. Tollip is a negative regulatory factor in TLRs signaling pathway, which can inhibit excessive and long-term immune response. SjTollip is a relatively conserved protein with homologous sequences in many species. The recombinant plasmid of pET-28a/SjTollip was constructed, and the insoluble protein was induced to express. The high concentration and purity of recombinant protein; Western blot were obtained by urea purification. The results showed that SjTollip had certain immunogenicity. The titer of polyclonal antibody of rabbit immunized with SjTollip reached 1: 256000 Sj Tollip with high transcription level in cercariae, cystis, eggs, juvenile worms, female worms, and low transcription levels in cercariae and males, and SjTollip was mainly expressed in juvenile and female worms, which was consistent with gene transcription. The sensitivity and specificity of rSjTollip were 70% and 80%, respectively. The worm reduction rate of rSjTollip immunized mice was 58.05%, and the fecal egg reduction rate of 40d-42d was 59.84 days after infection. The fecal egg reduction rate was 78.68 days after infection. It can induce strong immune protection and is a potential vaccine candidate target. SSX protein is a member of CT antigen family. The heterotopic expression of CT antigen can lead to the immune response of autologous uniform cells or humoral fluids, and can be used to construct and develop immunotherapy protocols. As a target of immunotherapy, the homologous sequence of SjSSX and Schistosoma japonicum host was low. The recombinant plasmid of pET-28a/SjSSX was constructed and induced to express insoluble protein. High concentration and purity of recombinant protein were obtained by urea purification. Western blot analysis showed that SjSSX had good immunogenicity, the titer of SjSSX immunized rabbit polyclonal antibody reached 1: 256000, and the transcription level of SjSSX was higher in chicks and females, and the transcription level was lower in cercariae, cercariae and male worms than in cystis, eggs, cercariae and males. SjSSX was expressed in egg, cercariae, child worm, male worm, female worm, SjSSX was used as coating antigen to detect serum of different population by ELISA method, sensitivity was 80, specificity was 90. Mice immunized with SjSSX produced 28.16% worm reduction rate and 4.25% egg reduction rate in 43d-45d feces after infection.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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