导　　师： 沈振国

学科专业： 071001

授予学位： 硕士

作　　者： ;

机构地区： 南京农业大学

摘　　要： 金属硫蛋白/(MT/)是一类分子量较小、富含Cys的金属结合蛋白,广泛分布于生物界。近年来研究表明,金属硫蛋白不仅参与体内微量元素的储存、转运和代谢,还具有拮抗电离辐射,清除自由基以及重金属解毒等作用。而植物金属硫蛋白的发现相对较晚,研究表明,植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧,使植物免受氧化损伤。它还参与了植物的发育、胚胎发生、抵抗逆境胁迫等多种生理过程,因此对植物的生长非常重要。 本研究旨在建立海州香薷金属硫蛋白的高效表达及纯化体系,制备其多克隆抗体。根据已克隆的海州香薷金属硫蛋白基因序列,设计并合成一对特异性PCR引物。以基因cDNA第一链为模板,通过PCR扩增获得MT基因,并进一步将之克隆在质粒载体pGEM-T easy上。用限制性内切核酸酶从该载体上切下目的基因,然后插入到表达载体pGEX-2T上获得重组表达质粒,转化大肠杆菌BL21,IPTG进行诱导表达,得到分子量约为33kDa的GST-EhMT1融合蛋白。该融合蛋白以可溶性和包涵体两种形式存在。可溶性蛋白经GST亲和树脂纯化后得到高纯度的目的蛋白。以融合蛋白作为抗原免疫家兔制备得到多克隆抗体。本实验取得如下研究结果： 1.成功地将扩增得到的目的基因EhMT1与克隆载体pGEM-T easy连接并转化到宿主菌JM109中,构建了重组克隆载体pGEM-T-EhMT1。 2.构建了EhMT1的原核表达载体pGEX-2T-EhMT1。序列分析表明,目的基因按正确的阅读框架插入到表达载体GST序列中。筛选出阳性重组体,表达的蛋白产物经SDS-PAGE证明EhMT1基因在大肠杆菌中成功得到了表达,其分子量约为33kDa. 3.经过优化目的融合蛋白的表达条件后,得到了含量比较高的可溶性融合蛋白。该可溶性融合蛋白纯化后得到单一的目的融合蛋白条带。 4.以目的融合蛋白作为抗原� Metallothioneins, which widely distribute in eukaryotic and prokaryotic organisms, are a class of cystein-rich and heavy metal-binding proteins with low molecular weight. Recently, many studies demonstrated that metallothioneins played a role in storing and carring trace elements, scavenging of free radicals and detoxifying of heavy metals. The study on plant MTs is just started because the discovery of plant MTs is relatively later. Many studies demonstrated that the plant metallothionein may function in both metal chaperoning and scavenging of reactive oxygen species with their large number of cysteine residues to protect plants from oxidative damages and participated in many physiological proeesses, such as development of plant, embryogenesis and stress resistance, and was regarded as an important functional gene. As a result, Metallothioneins exert quite significant function in process of plants remediation. According to the sequences of EhMT1 gene cloned, a pair of special primers were designed and synthesized, we obtained coding sequence of EhMT1 from cDNA by polymerase chain reaction/(PCR/), and further cloned it into the pGEM-T easy vector. The fragment was again inserted into the prokaryotic expression vector pGEX-2T when it was cut out with two restrictionenzymes. Then, the recombinant plasmid pGEX-2T-EhMT1 was transferred into E.coli BL21. After being induced by IPTG, GST-EhMT1 fusion protein, who's relative molecular mass is 33kDa, was expressed in two different kinds of forms-soluble protein and inclution body. High purity fusion protein was obtained after the soluble protein was purified by the GST-tag affinity chromatography. The purified fusion protein GST-EhMT1 was injected into rabbits, the polycloned antibody was produced. The results are as follows: 1 Recombinant cloning vector of pGEM-2T-EhMT1 was successfully constructed by ligating the EhMT1 gene into pGEM-T easy vector and transformed into JM109. 2 Recombinant prokaryotic expression vector was successfully construc

关 键 词： 金属硫蛋白 原核表达 融合蛋白 抗血清

领　　域： [生物学]