人tau蛋白外显子2和外显子3特异性抗体的制备  

Preparations of the specific antibodies against exon 2 and exon 3 of human tau protein

作　　者：陈操[1] 王桂荣[2] 石琦[2] 张宝云[2] 梅国勇[2] 李媛[2] 王新[2] 周瑞敏[2] 韩俊[2] 赵玉军[1] 董小平[2] 

机构地区：[1]沈阳农业大学畜牧兽医学院,110161 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室

出　　处：《中华实验和临床病毒学杂志》2009年第2期146-148,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：基金项目：国家自然科学基金委项目（30771914、30571672、30500018、30800975）;“973”计划（2007CB310505）;科技支撑项目（2006BAD06A13.2）;科研院所基金资助项目（2008EG150300）

摘　　要：目的制备人微管相关蛋白tauN端的外显子2和外显子3特异性多克隆抗体。方法从人外周血DNA中获得tau蛋白外显子2及外显子3序列并连接至原核表达载体pGEX-2T，表达纯化重组融合蛋白GST—E2、GST-E3；以此融合蛋白免疫家兔及小鼠制备抗血清，通过ProteinG／A、偶联GST蛋白的溴化氰（CNBr）活化柱对抗血清进行亲和纯化；用WesternBlot及ELISA方法确定所制备抗体的特异性和敏感性。结果在大肠埃希菌中表达纯化出人tau外显子2和外显子3重组融合蛋白GST-E2和GST-E3，相对分子质量为29×10^3。免疫实验动物后获得抗血清，经系列纯化后WesternBlot和ELISA检测显示，制备的抗人tau蛋白外显子2和外显子3抗体具有良好的特异性和免疫反应效价。结论成功制备了4种人tau外显子2和外显子3特异性抗体，为进行tau蛋白在神经退行性疾病中的作用研究提供了基础。Objective To prepare the specific antibodies against exon 2 and exon 3 of human tau protein. Methods Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA. Results Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 ×10^3 . Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes. Conclusion Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.

关 键 词：蛋白质类 外显子 抗体 病毒 朊病毒病 

分 类 号：R392[医药卫生—免疫学]