【作者】 方堃；

【导师】 陈玉林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 山羊绒的主要组成成分是角蛋白,角蛋白同时又是多种氨基酸的组合体,其中含量最高的是谷氨酸和胱氨酸,胱氨酸是含硫氨基酸。羊等哺乳动物自身不能直接合成半胱氨酸,只能依靠反式硫化蛋氨酸间接的合成半胱氨酸。利用转基因技术,将半胱氨酸合成酶转移至绒山羊体内,使得羊体自身能够高效的合成半胱氨酸,对山羊绒品质的提高有极大促进作用。本研究旨在通过PCR扩增大肠杆菌(Escherichia coli)丝氨酸乙酰转移酶基因(cysE)和O-乙酰丝氨酸硫化氢解酶基因(cysM),构建质粒载体进行原核表达,并制备相应的蛋白多克隆抗体,为生产半胱氨酸合成酶转基因绒山羊奠定方法与技术基础。(1)从大肠杆菌基因组DNA中扩增cysE基因,长度为822 bp,编码274个氨基酸残基,cysM基因全长912 bp,编码304个氨基酸残基,各为1个完整的开放阅读框。测序后得到的序列,与Genbank公布的cysE(8177668)和cysM(8176794)序列相比对,同源性达到100%。经基因重组成功构建原核表达载体pET32a-cysE和pET32a-cysM,并可在大肠杆菌BL21(DE3)中诱导后实现高效表达。(2)原核表达载体pET32a-cysE和pET32a-cysM在大肠杆菌BL21(DE3)经1 mmol/L IPTG诱导,37℃下,表达6 h,目的蛋白表达量超过全部菌蛋白表达量的25%。通过Ni-Agrose亲和柱纯化,从细菌裂解上清液中纯化得到了cysE和cysM融合蛋白。SDS-PAGE结果显示,亲和柱纯化法得到的融合蛋白纯度可达90%,回收效率达60%。(3)用纯化的cysE和cysM蛋白作为抗原免疫新西兰大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,抗体滴度分别达到1:102.4K,表明纯化制备的多克隆抗体具有较强的免疫结合活性。同时,以兔血清为一抗,HRP标记的羊抗兔IgG为二抗,对得到的cysE和cysM融合蛋白进行Western blot检测,表明试验获得抗体对原核表达的cysE和cysM融合蛋白具有特异性反应。通过本研究建立了大肠杆菌cysE和cysM基因的克隆、原核表达及多克隆抗体制备的方法与技术。为进一步研究半胱氨酸合成酶转基因绒山羊奠定了技术基础,具有重要理论与育种实践价值。更多还原

【Abstract】 The major component of goat cashmere fiber is keratin, which is a variety of combination of amino acids. The highest contents of keratin are glutamic acid and cystine, and cystine is a sulfur-containing amino acids. Goat and other mammals can only synthesis cysteine indirectly from methionine viatrans-sulphuration. Using genetic engineering, the expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the goat. It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. This study focuses on cloning serine acetyltransferase gene (cysE) and O-acetylserine sulfhydrylase-B gene (cysM) which encoding cysteine synthetase in Escherichia coli, prokaryotic expressing the enzymes and preparing their antibodies, which as preliminary works for the production of transgenic cashmere goat.(1) The cysE and cysM genes are amplified by PCR from Escherichia coli genomic DNA. The cysE gene is 822 bp, which encoding 274 amino acids, and cysM gene is 912 bp, which encoding 304 amino acids. Each one of them is a complete open reading frame. Sequences alignment between sequencing results and published Genbank cysE (8177668) and cysM (8176794) sequences shows 100% homology. Prokaryotic expression vectors pET32a-cysE and pET32a-cysM are successfully constructed and efficiently express in BL21 (DE3) after induced.(2) Induce by 1 mmol/L IPTG, 37℃, 6h, prokaryotic expression vectors pET32a-cysE and pET32a-cysM efficiently express in Escherichia coli BL21 (DE3), the expression of target protein over all the bacterial protein by 25%. The cysE and cysM proteins are purified from Bacterial lysate by Ni-Agrose affinity purification. SDS-PAGE results show that over 60% of the proteins are recovered, and the purity of purified proteins is 90%.(3) Using purified cysE and cysM proteins as antigens immunize New Zealand white rabbits to create polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) detection shows that antibodies titers reach 1:102 400, indicates polyclonal antibodies have a strong immune binding activity. At the same time, using rabbit serum as primary antibody, HRP labeled goat anti-rabbit IgG as secondary antibody, the Western blott result shows that the antibodies have specific responses to cysE and cysM proteins. Cloning, prokaryotic expression and polyclonal antibody preparation methods and techniques of Escherichia coli cysE and cysM gene were successfully established. Identified and purified cysE and cysM protein from prokaryotic expression. The preparation of recombinant cysE, cysM and their polyclonal antibodies has provided reliable tools for the future study in the transgenic sheep of cysteine biosynthesis gene.更多还原