本文選題：豬帶絳蟲　切入點(diǎn)：組織蛋白酶B　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：豬囊尾蚴能夠引起豬和人的囊蟲病,影響?zhàn)B豬業(yè)的發(fā)展和人類的健康。組織蛋白酶B是半胱氨酸蛋白酶家族成員,在寄生蟲的營養(yǎng)、生長發(fā)育、組織遷移、蛋白加工和活化以及免疫逃避等方面發(fā)揮重要作用,是預(yù)防及治療多種寄生蟲疾病的潛在的藥物靶標(biāo)和疫苗候選分子。本文對豬帶絳蟲組織蛋白酶B基因進(jìn)行了克隆、表達(dá)及功能的初步研究。取得了以下結(jié)果:1.以豬帶絳蟲的c DNA為模板,成功地?cái)U(kuò)增了豬帶絳蟲組織蛋白酶B基因(Tscp B)。利用生物信息學(xué)工具,對該序列進(jìn)行分析。結(jié)果表明該基因ORF大小為1074 bp,編碼357個(gè)氨基酸,理論分子質(zhì)量為39.27 ku,具有組織蛋白酶B的特征結(jié)構(gòu)域。Real-time PCR結(jié)果表明,組織蛋白酶B基因在成蟲期表達(dá)量顯著高于囊尾蚴時(shí)期。2.構(gòu)建了原核表達(dá)重組質(zhì)粒p ET30a-Tscp B,經(jīng)誘導(dǎo)表達(dá),獲得了以包涵體形式存在的重組蛋白r Tscp B,分子大小約為38 ku。Western blotting分析表明,r Tscp B能被豬囊尾蚴病陽性血清特異性識(shí)別。用純化后的r Tscp B免疫家兔,4次免疫后產(chǎn)生了特異性的高滴度Ig G抗體。免疫組化結(jié)果表明,r Tscp B主要表達(dá)于成蟲的體壁及生殖器官,證明豬帶絳蟲組織蛋白酶B可能與蟲體攝取營養(yǎng)以及生殖發(fā)育有關(guān)。3.構(gòu)建了畢赤酵母真核表達(dá)重組質(zhì)粒p PIC9K-Tscp B。明膠酶譜實(shí)驗(yàn)證明y Tscp B具有水解活性,且能夠降解熒光底物Z-Phe-Arg-AMC,最適p H為5.5。y Tscp B還能降解豬Ig G、BSA、纖連蛋白等大分子。y Tscp B的水解活性能被E-64特異性抑制。底物實(shí)驗(yàn)表明豬帶絳蟲組織蛋白酶B可能在攝取營養(yǎng)、入侵宿主等方面發(fā)揮作用。4.利用同源模建的方法預(yù)測了豬帶絳蟲組織蛋白酶B蛋白的結(jié)構(gòu),利用分子對接的方法預(yù)測了組織蛋白酶B蛋白與抑制劑E-64之間相互作用的模式。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 陳焱,易新元,曾憲芳,汪世平;日本血吸蟲成蟲31/32kD抗原誘導(dǎo)小鼠產(chǎn)生抗卵免疫的初步研究[J];中國血吸蟲病防治雜志;1995年02期

2 沈定文,李雍龍,韓家俊,石佑恩;日本血吸蟲成蟲31/32 kDa蛋白的純化及保護(hù)性免疫力的研究[J];中國寄生蟲學(xué)與寄生蟲病雜志;1993年04期

3 張京,張中庸,李靚如,馬春艷,吳蓮英;豬囊尾蚴病免疫治療研究[J];中國人獸共患病雜志;2000年06期

相關(guān)碩士學(xué)位論文 前1條

1 李君君;豬帶絳蟲半胱氨酸蛋白酶抑制劑基因家族的鑒定[D];中國農(nóng)業(yè)科學(xué)院;2014年

，

本文編號(hào)：1694212