热休克蛋白90α(HSP90α)在大肠杆菌中的表达  

Expression of HSP90α in E. coli

作　　者：乔绘红[1] 赵玫[2] 施一江[2] 袁兴华[2] 范云霞[2] 周纯[2] 徐枫[2] 黄常志[2] 

机构地区：[1]北京市东单三条5号医科院基础所病理室,北京100005 [2]中国医学科学院中国协和医科大学肿瘤研究所,北京100021

出　　处：《中国免疫学杂志》2000年第3期119-121,共3页Chinese Journal of Immunology

基　　金：国家自然科学基金!资助项目 (批准号 3 9770 82 4)

摘　　要：目的 :探讨热休克蛋白 90α的生物学功能 ,获得高质量及充足的HSP90α蛋白。方法 :采用PCR的方法扩增HSP90αcDNA5’端 42 4bp片段 ,在起始密码子位置制造一个NcoI酶切位点 ,将PCR产物克隆到中间载体 ,再通过合适的酶切位点将其余片段连接到此载体 ,形成一个带有NcoI突变位点的HSP90αcDNA全长克隆。最终将HSP90α全部编码cDNA插入到pET 16b表达载体中。结果 :经诱导表达 ,SDS PAGE凝胶电泳分离显示在分子量大约 83kD处有一诱导蛋白带。WesternBlot证明表达产物与抗HSP90α抗血清有特异的免疫反应。结论 :构建成了T7启动子控制下的HSP90α表达质粒。Objective:To acquire high quality HSP90α protein.Methods:Hsp90α cDNA 5' end 424 bp fragment was amplified by PCR.Three bases were mutated,so NcoI restriction enzyme site was produced.The PCR product was successively inserted into intermediate vector,by some restriction enzyme hsp90α 3' end fragment was inserted into the vector.Thus,a clone was obtained which contains whole fragment of hsp90α cDNA with NcoI restriction enzyme site.After cleaved with NcoI and BamHI,the hsp90α cDNA was cloned into pET 16 blxpression vector.Results:tSDS PAGE gel electrophoresis showed that a protein band with molecular weight of 83 kD appeared as the expected size after transformation and inductionin the host bacteria (DE3).Western blot analysis showed that the expressed protein was hybridized with the antibody to HSP90α. Conclusion:HSP90α expressing plasmid was constructed.

关 键 词：热休克蛋白 大肠杆菌 肿瘤 免疫方法 

分 类 号：R730.51[医药卫生—肿瘤] Q784[医药卫生—临床医学]