Us11的表达及其多克隆抗体的制备

doi:10.13560/j.cnki.biotech.bull.1985.2016.03.033

生物技术通报 ›› 2016, Vol. 32 ›› Issue (3): 209-214.doi: 10.13560/j.cnki.biotech.bull.1985.2016.03.033

Us11的表达及其多克隆抗体的制备

童海燕¹,李国毅^1,2,汤颖¹

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

收稿日期:2015-05-28 出版日期:2016-03-24 发布日期:2016-03-25
作者简介:童海燕, 硕士研究生, 研究方向：中枢神经系统感染；E-mail：tonghaiyan1215@163.com
基金资助:
黑龙江省自然科学基金项目(D201219)

Expression of Protein Us11 and Preparation of Polyclonal Antibodies

TONG Hai-yan¹, LI Guo-yi^{1, 2}, TANG Ying¹

1. The First Affiliated Hospital of Harbin Medical University, Harbin 150001；
2. Oriental Hospital Affiliated to Tongji University, Shanghai 200085

Received:2015-05-28 Published:2016-03-24 Online:2016-03-25

摘要/Abstract

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pCold-Us11和pFLAG-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pFLAG-Us11转染293T细胞表达的Us11蛋白。

关键词: Us11, 原核表达, 真核表达, 多克隆抗体

Abstract: 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 293T cells；the expressed protein was detected by indirect immunofluorescence(IFA)and Western blot(WB). Plasmid pCold-Us11 and pFLAG-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 283T cells by pFLAG-Us11.

Key words: Us11, prokaryotic expression, eukaryotic expression, polyclonal antibody

童海燕,李国毅,汤颖. Us11的表达及其多克隆抗体的制备[J]. 生物技术通报, 2016, 32(3): 209-214.

TONG Hai-yan, LI Guo-yi, TANG Ying. Expression of Protein Us11 and Preparation of Polyclonal Antibodies[J]. Biotechnology Bulletin, 2016, 32(3): 209-214.

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Us11的表达及其多克隆抗体的制备

Expression of Protein Us11 and Preparation of Polyclonal Antibodies

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