人GlnRSN端多肽的原核表达、纯化及其多抗的制备  

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of N-terminal Polypeptide of Human GlnRS Protein

作　　者：解帅帅[1] 韩宏岩[1] 许维岸[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,苏州215123

出　　处：《基因组学与应用生物学》2016年第10期2597-2602,共6页Genomics and Applied Biology

基　　金：国家自然科学基金项目(No.81071306)资助

摘　　要：制备谷氨酰胺-t RNA合成酶(Gln RS)多克隆抗体,为下一步深入研究其结构与功能做准备。以Gln RS全长基因为模板,PCR获取编码Gln RS N端236个氨基酸的基因片段,将PCR产物插入p ET28a以构建N(1-236)-Gln RS的重组表达载体(p ET28a-N-Gln RS),转化大肠杆菌BL21(DE3)进行诱导表达N-Gln RS重组蛋白,用镍柱对表达的蛋白进行纯化,将纯化的蛋白对BALB/c小鼠采取皮下注射制备多克隆抗体,离心取血清,最后对制备的抗体进行特异性检测。PCR获得了目的片段,成功构建表达载体,获得表达的Gln RS N端236个氨基酸重组蛋白,制备抗体具有很好的特异性,可用于Gln RS检测。以N-Gln RS重组蛋白为抗原制备的抗体对Gln RS具有很好的免疫特异性,为下一步在蛋白水平深入研究Gln RS的结构与功能提供了抗体准备。The experiment is conducted to obtain the protein of N-terminal polypeptide of GlnRS from prokaryo- tic cells and parapre polyclonal antibody against GlnRS. The gene of N-terminal Polypeptide of GlnRS gene was amplified by PCR from pET28a-GInRS plasmid contained full lenth cDNA of GInRS and was cloned into pET28a between EcoR I and Xho I to construct expression vector. The constructed plasmid was inserted into BL21 to express protein. Protein expression was induced by IPTG in BL21 and the expressed protein was purified by NI-NTA affinity chromatography. Subsequently, BALB/c mice were injected subcutaneously with purified proteins and the specificity of the antiserums was assayed by western blot. The expression vector was constructed successfully and the protein was expressed efficiently in BL21. The western blot results demonstrate that the obtained polyclonal antibody can be used in full lenth GlnRS assay with high specificity. The obtained polyclonal antibody shows high specificity and lays foundations for the study of structure and function ofGlutaminyl-tRNA synthetase.

关 键 词：谷氨酰胺-tRNA合成酶 载体构建 原核表达 多克隆抗体制备 

分 类 号：Q78[生物学—分子生物学]