代谢型谷氨酸受体5的纯化及纳米抗体的筛选  

Purification of Metabolic Glutamate Receptor 5 and Screening of Nanoantibodies

作　　者：王辉[1,2,3] 马梦洁 许剑锋 WANG Hui;MA Mengjie;XU Jianfeng(Food College,Shanghai Ocean University,Shanghai 201306,China;Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201306,China;National Experimental Teaching Demonstration Center for Food Science and Engineering(Shanghai Ocean University),Shanghai 201306,China)

机构地区：[1]上海海洋大学,上海201306 [2]中国科学院上海药物研究所,上海201203 [3]食品科学与工程国家级实验教学示范中心(上海海洋大学),上海201306

出　　处：《工业微生物》2023年第5期156-162,共7页Industrial Microbiology

基　　金：国科金外资助字第(81750110548)号。

摘　　要：文章首先采用细胞表达抑制状态的代谢型谷氨酸受体5(Metabotropic glutamate receptor 5,m GluR5)蛋白免疫野生双峰驼,构建纳米抗体文库,运用M13噬菌体展示技术,筛选出与m Glu R5具有特异性结合的纳米抗体;其次用细胞对m GluR5蛋白进行表达纯化,然后对野生单峰驼进行7次免疫,对抽取骆驼的外淋巴细胞总RNA进行提取,经过反转录的方式转为c DNA,将用巢式PCR对VHH片段进行扩增并与p MECS噬菌粒载体进行连接,并电转化至TG1大肠杆菌中构建纳米抗体噬菌体展示文库;其三通过M13噬菌体展示技术对文库进行淘选,淘选出多个互补决定区3序列不同的纳米抗体;最后纯化出纯度较高的m GluR5蛋白,构建出库容量为5.47×10^(8)CFU/mL的噬菌体文库菌,经过两轮液相淘选,获得了12个互补决定区3序列不同的纳米抗体。研究成功筛选出12个与m GluR5特异性结合较高的纳米抗体,为m Glu R5的靶向药物研发和G蛋白偶联受体的信号通路研究奠定了基础。Wild Bactrian camel was immunized with metabotropic glutamate receptor 5(mGluR5)protein expressed in cells,and a library of nano-antibodies was constructed.The nano-antibodies specifically bound to mGluR5 were screened by M13 phage display technology.The mGluR5 protein was expressed and purified by cells,and then wild dromedan camels were immunized seven times.The total RNA of extracellular lymphocytes of camels was extracted and converted into cDNA by reverse transcription.Nested PCR was used to amplify VHH fragment and connect it with pMECS granula carrier,and it was electrically transformed into TG1 Escherichia coli to build a phage library of nano antibody display.The library was panned by M13 phage display technology,and several antibodies with different sequences of complementary determinate region 3 were panned out.High purity mGluR5 protein was purified,and a phage library bacterium with a library capacity of 5.47×10^(8)CFU/ml was constructed.After two rounds of liquid phase panning,12 nanoantibodies with different sequences of complementary determinate region 3 were obtained.12 nano antibodies with high specific binding to mGluR5 were successfully screened,which laid a foundation for the development of mGluR5 targeting drugs and the study of G protein-coupled receptor signaling pathway.

关 键 词：代谢型谷氨酸受体5 M13噬菌体展示技术 纳米抗体 

分 类 号：TQ460.1[化学工程—制药化工] TB383.1[一般工业技术—材料科学与工程]