抗热休克蛋白72多克隆抗体血清的制备和检测  

Preparation and identification of polyclonal antiserum against heat shock protein 72

作　　者：黄帆[1] 徐康[2] 吴兴刚[2] 马中富[3] 林正[2] 

机构地区：[1]中山大学附属第一医院神经内科,广州510080 [2]中山大学附属第一医院中心实验室,广州510080 [3]中山大学附属第一医院普内科,广州510080

出　　处：《中国危重病急救医学》2006年第9期531-534,共4页Chinese Critical Care Medicine

基　　金：国家自然科学基金资助项目(30070829);广东省自然科学基金资助项目(5001731)

摘　　要：目的探讨在大肠杆菌中表达人热休克蛋白72(hHSP72)与组氨酸(His)的融合蛋白并制备其抗体血清的方法。方法将hHSP72片断插入His融合表达载体pPROEX1TM,重组载体酶切鉴定后,在大肠杆菌中经异丙基βD硫代半乳糖苷(IPTG)诱导表达获得His hHSP72融合蛋白。采用纯化后的HSP72免疫新西兰白兔,制备抗血清;蛋白质免疫印迹法(Western blot)检测重组抗原的免疫活性。结果重组质粒酶切鉴定结果表明,hHSP72基因已正确插入到pPROEX1TM中,经IPTG诱导后,表达出分子质量约为73ku的蛋白,获得了效价为1∶100000的多克隆抗体。Western blot检测证明,所制备的多克隆抗体可以与hHSP72特异性结合,其效价高于市售的单克隆抗体。结论用hHSP72片断在大肠杆菌中能成功表达,并能制备得到多克隆抗体;这种多克隆抗体是一种新的高特异性和高灵敏度的试剂。Objective To express fusion protein of histamine （His） and human heat shock protein 72 （hHSP72） in Escherichia coli （E. coli）, and to prepare hHSP72 antiserum in rabbit. Methods hHSP72 gene was inserted into pPROEX - 1^TM. The recombinant vector was identified by restriction endonuclease digestion analysis and sequence. Fusion protein His -hHSP72 was expressed in E. coli under isopropy- β- D- thiogalactoside （IPTG） induction. The rabbit antibody against His -hHSP72 was prepared by using purified His -hHSP72 protein as immunogen, and the specificity and sensitivity of polyclonal antibody were identified by Western blot. Results The restriction endonuclease digestion analysis of recombinant plasmid and sequence demonstrated that the hHSP72 gene had been exactly inserted into pPROEX -1^TM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS- PAGE） analysis showed that the relative molecular mass of the fusion protein was about 73 ku. Western blot result proved that the rabbit polyclonal antiserum could fuse with over 20 ng hHSP72 protein when diluted to 1 ： 100 000. Conclusion The polyclonal antibody against hHSP72 can be prepared in E. coli, it is a new reagent with high specificity and sensitivity for the research of hHSP72.

关 键 词：热休克蛋白72 载体构建 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]