猪带绦虫组织蛋白酶B基因的克

发布时间：2018-04-01 05:32

本文选题：猪带绦虫　切入点：组织蛋白酶B　出处：《中国农业科学院》2015年硕士论文

【摘要】：猪囊尾蚴能够引起猪和人的囊虫病,影响养猪业的发展和人类的健康。组织蛋白酶B是半胱氨酸蛋白酶家族成员,在寄生虫的营养、生长发育、组织迁移、蛋白加工和活化以及免疫逃避等方面发挥重要作用,是预防及治疗多种寄生虫疾病的潜在的药物靶标和疫苗候选分子。本文对猪带绦虫组织蛋白酶B基因进行了克隆、表达及功能的初步研究。取得了以下结果:1.以猪带绦虫的c DNA为模板,成功地扩增了猪带绦虫组织蛋白酶B基因(Tscp B)。利用生物信息学工具,对该序列进行分析。结果表明该基因ORF大小为1074 bp,编码357个氨基酸,理论分子质量为39.27 ku,具有组织蛋白酶B的特征结构域。Real-time PCR结果表明,组织蛋白酶B基因在成虫期表达量显著高于囊尾蚴时期。2.构建了原核表达重组质粒p ET30a-Tscp B,经诱导表达,获得了以包涵体形式存在的重组蛋白r Tscp B,分子大小约为38 ku。Western blotting分析表明,r Tscp B能被猪囊尾蚴病阳性血清特异性识别。用纯化后的r Tscp B免疫家兔,4次免疫后产生了特异性的高滴度Ig G抗体。免疫组化结果表明,r Tscp B主要表达于成虫的体壁及生殖器官,证明猪带绦虫组织蛋白酶B可能与虫体摄取营养以及生殖发育有关。3.构建了毕赤酵母真核表达重组质粒p PIC9K-Tscp B。明胶酶谱实验证明y Tscp B具有水解活性,且能够降解荧光底物Z-Phe-Arg-AMC,最适p H为5.5。y Tscp B还能降解猪Ig G、BSA、纤连蛋白等大分子。y Tscp B的水解活性能被E-64特异性抑制。底物实验表明猪带绦虫组织蛋白酶B可能在摄取营养、入侵宿主等方面发挥作用。4.利用同源模建的方法预测了猪带绦虫组织蛋白酶B蛋白的结构,利用分子对接的方法预测了组织蛋白酶B蛋白与抑制剂E-64之间相互作用的模式。
[Abstract]:Cysticercosis can cause cysticercosis in pigs and humans, and affect the development of pig industry and human health. Cathepsin B is a member of the cysteine protease family, which is responsible for the nutrition, growth and tissue migration of parasites. Protein processing and activation as well as immune evasion play an important role as potential drug targets and vaccine candidates for the prevention and treatment of various parasitic diseases. The cathepsin B gene of Taenia solium was cloned in this paper. Using c DNA of Taenia solium as template, the cathepsin B gene of Taenia solium was successfully amplified by using bioinformatics tools. The results showed that the ORF size of the gene was 1074 BP, encoding 357 amino acids, and the theoretical molecular weight was 39.27 ku.Real-time PCR showed that the gene had the characteristic domain of cathepsin B. The expression of cathepsin B gene in adult stage was significantly higher than that in cysticercaria stage. The recombinant protein r Tscp B in the form of inclusion body was obtained. The molecular size of r Tscp B was about 38 ku.Western blotting. The results showed that r Tscp B could be specifically recognized by the positive serum of cysticercosis. The purified r Tscp B was used to immunize rabbits for 4 times. Specific high titer IgG antibody was produced. The immunohistochemical results showed that Tscp B was mainly expressed in the body wall and reproductive organs of adults. The results showed that cathepsin B of Taenia solium might be related to the uptake of nutrients and reproductive development of P. solium. The recombinant plasmid p PIC9K-Tscp B. Gelatinase spectrum of Pichia pastoris was constructed. The result of gelatinase spectrum showed that y Tscp B had hydrolytic activity. The biodegradation of fluorescent substrate Z-Phe-Arg-AMCs, the optimum pH value of 5.5.y Tscp B, and the degradation of porcine Ig GfB, fibronectin and other macromolecules. The hydrolysis activity of the macromolecule. Y Tscp B was specifically inhibited by E-64. The substrate experiments showed that cathepsin B of Taenia solium might be uptake of nutrients. The homologous modeling method was used to predict the structure of cathepsin B protein of Taenia solium and the interaction pattern between cathepsin B protein and inhibitor E-64 was predicted by molecular docking method.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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