重组小鼠14-3-3蛋白theta亚型的构建

目的克隆编码14—3—3蛋白质θ亚型的cDNA,制备该蛋白质的基因重组蛋白质．方法参照小鼠14—3-3蛋白质θ亚型mRNA结构设计引物,以小鼠脑总RNA为模板,RT—PCR法克隆编码14—3—3蛋白质θ亚型的cDNA,插入原核表达质粒pGEX-4T-1,转化大肠杆菌BL21（DE3）使其表达谷胱甘肽S转移酶（GST）为标签的融合蛋白质,以凝血酶定点分解融合蛋白并采用GlutathioneSepharose4B亲和层析纯化目的蛋白．结果RT—PCR扩增产物经1％琼脂糖凝胶电泳分离可见约800bp条带,序列分析表明重组质粒pGEX-1433theta中的插入子为738bp,编码245个氨基酸,其核苷酸及氨基酸序列与PubmedNM-011739展示的结果一致．重组质粒pGEX-1433theta在BL21（DE3）中的表达量随IPTG诱导时间递增,融合蛋白质为可溶性组分．亲和层析纯化的14—3—3theta在SDS．PAGE上表现为单一条带,表观相对分子质量为30ku．每L培养菌液可收获4mg基因重组型小鼠14—3—3theta．结论克隆了编码小鼠14—3-3蛋白质θ亚型的cDNA,制备了高纯度基因重组型小鼠14—3—3theta,为进行单克隆抗体的制备奠定了坚实的基础．

Construction of Recombinant Mouse 14-3-3 Protein Theta

Objective Cloning the eDNA of 14-3-3θ protein and making its gene recombinant protein. Method According the mRNA structure of mouse 14-3-3θ, we designed the primer. The cDNA fragments encoding the 14-3-3θ were cloned from mouse brain by RT-PCR,and cloned into pGEX-4T-1. The recombinant plasmids pGEX-14-3-3θ were transformed into the bacteria BL2.1 （DE3）, and positive clones were induced with IPTG to express （GST） target protein and characterized by SDS-PAGE. Purified by Glutathione Sepharose 4B affinity column chromatograph and cleaved with thrombin. Results The size of fragment RT-PCR products was about 800bp by SDS-PAGE analysis. Sequence analysis showed that the size of the recombinant plasmid was 738bp, coding 245 amino acid. The sequence of nucleotide acid and amino acid of inserts in positive clones were coincident with the original sequence of 14-3-3θ gene from Pubmed NM-011739. The recombinant expression plasmids were constructed through sub-cloning the right inserts of 14-3-3θ into pGEX-4T-1. The fusion protein was soluble. The purified 14-3-3θ protein by affinity column chromatograph appears only one strap. The molecular weight was 30ku. We can get 4mg gene recombinant mouse 14-3-3theta per liter bacterium. Conclusion Cloning the cDNA of 14-3-3θ protein and making highly purified gene recombinant protein. Making firm base for the preparation of monoclonal antibody.