Prokaryotic recombiant expression,purification and polyclonal antibody preparation of extracellular domain of programmed cell death 1 ligand 1

摘要：

为制备程序性细胞死亡配体1胞外区多肽及其鼠源性多克隆抗体,根据UniProtKB数据库中公布的其胞外区基因序列(标识符:Q9NZQ7-1)和原核表达载体pET-28a(+)Nde I上游和Xho I位点下游的序列,设计合成带有同源臂的特异性引物.以合成的PD-L1胞外区基因为模板,PCR扩增程序性细胞死亡配体1胞外区基因并将其克隆至pET-28a(+)中,构建重组质粒pET-28a(+)-PD-L1-E,转化至大肠杆菌DH5α中,扩增并提取其质粒,然后将质粒转化至大肠杆菌BL21(DE3)中进行诱导表达.亲和层析镍柱纯化重组蛋白后,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离飞行时间质谱鉴定,确认表达蛋白正确.浓缩换液后用重组蛋白免疫Balb/c品系小鼠制备多克隆抗体,经ELISA检测,成功获得抗程序性细胞死亡配体1胞外区血清.该研究对于研发某些肿瘤伴随诊断检测试剂有重要意义.

In order to prepare the polypeptides of extracellular domain of programmed cell death 1 ligand 1(PD-L1)and its mouse-derived polyclonal antibody,we designed and synthesized the specific primers with homologous arms according to the gene sequence of its extracellular domain published in UniProtKB(identifier:Q9NZQ7-1)and the Nde I upstream and Xho I downstream sequences of the prokaryotic expressive vector pET-28a(+).The artificially synthesized gene encoding the extracellular domain of PD-Ll as a template was amplified by PCR.The amplified gene was cloned into the vector pET-28a(+)to construct the recombinant plasmid pET-28a(+)-PD-L1-E.The constructed plasmid was transformed into Escherichia coli DH5α,amplified and extracted.Then the extracted plasmid was transformed into E.coli BL21(DE3)to induce expression.The recombinant protein was purified by Ni-affinity chromatography,and then identified by SDS-PAGE and MALDI-TOF-MS/MS.After ultrafiltration concentration and buffer exchange,the recombinant protein was used to immunize Balb/c mice to prepare the polyclonal antibody against PD-L1.The antisera was successfully obtained by ELISA determination.The research has great significance for development of tumor companion diagnostic reagent.

作者：

薛德明 梁永波 常秋霜 翟晋豫 张东辉 柴素真 齐华

ligandXue Deming;Liang Yongbo;Chang Qiushuang;Zhai Jinyu;Zhang Donghui;Chai Suzhen;Qi Hua(College of Life Sciences,Henan Normal University,Xinxiang 453007,China;Research Center of Engineering Technology on Diagnostic Reagent of Tumor Pathology of Henan Province,Henan Celnovte Biotechnology Co.,Ltd.,Zhengzhou 450008,China)

机构地区：

betway官方app 生命科学学院 河南赛诺特生物技术有限公司河南省肿瘤病理诊断试剂工程技术研究中心

出处：

《betway官方app 学报：自然科学版》 CAS 北大核心 2020年第5期99-104,共6页

基金：

河南省国际科技合作计划项目(152102410038) 河南省重大科技专项(191110311200) 郑州市重大科技创新专项项目(188PCXZX789).

关键词：

程序性细胞死亡配体1胞外区 原核表达 多克隆抗体

extracellular domain of programmed cell death 1 ligand 1 prokaryotic expression polyclonal antibody

分类号：

Q812 [生物学—生物工程]

程序性细胞死亡配体1胞外区原核重组表达、纯化及其多克隆抗体制备.pdf