Us11的表达及其多克隆抗体的制备  

Expression of Protein Us11 and Preparation of Polyclonal Antibodies

作　　者：童海燕[1] 李国毅[1,2] 汤颖[1] 

机构地区：[1]哈尔滨医科大学第一附属医院,哈尔滨150001 [2]同济大学附属东方医院,上海200085

出　　处：《生物技术通报》2016年第3期209-214,共6页Biotechnology Bulletin

基　　金：黑龙江省自然科学基金项目(D201219)

摘　　要：已有研究发现单纯疱疹病毒1(HSV-1)感染后,病毒的Us11蛋白在拮抗宿主先天性免疫反应中发挥重要作用。通过合成HSV-1的Us11基因,克隆表达制备针对Us11的多克隆抗体,为研究Us11的功能以及与宿主蛋白的相互作用提供的实验基础。通过人工合成Us11基因并以其为模板扩增,回收Us11基因片段,克隆至原核和真核表达载体;诱导表达Us11蛋白并纯化,制备Us11兔源多克隆抗体;转染质粒至293T细胞,对细胞表达的蛋白进行间接免疫荧光(IFA)和免疫印迹(WB)检测。成功构建p Cold-Us11和p FLAG-Us11质粒,实现可溶性Us11蛋白表达,纯化的Us11蛋白免疫动物获得针对Us11的多克隆抗体。IFA和Western blot结果显示,制备的抗血清能特异性检测到p FLAG-Us11转染293T细胞表达的Us11蛋白。The previous studies have discovered that protein Us11 of HSV-1（herpes simplex virus 1）played an important role in antagonistic host innate immune responses after HSV-1 infection. By synthesizing the gene Us11 of HSV-1,the polyclonal antibodies for Us11 were cloned and prepared,which provided the solid experimental basis for studying the functions of Us11 and the interactions between Us11 and host protein. The artificially-synthesized gene Us11 was as template and amplified,and then the recycled gene fragments of Us11 were cloned into the prokaryotic and eukaryotic expression vectors. Further,the induced expressed Us11 protein was purified,and polyclonal antibodies for rabbit were prepared. The recombinant plasmid was transfected into 293 T cells;the expressed protein was detected by indirect immunofluorescence（IFA）and Western blot（WB）. Plasmid p Cold-Us11 and p FLAG-Us11 were successfully constructed,soluble Us11 protein expression was achieved,and the polyclonal antibodies targeting for Us11 was acquired by immunizing animal with purified Us11 protein. The results from IFA and WB revealed that prepared antiserum may specially detect the Us11 protein expressed in transfected 283 T cells by p FLAG-Us11.

关 键 词：Us11 原核表达 真核表达 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]