摘要
目的:构建小鼠白细胞介素-1α原核表达载体,表达并纯化IL-1α蛋白,制备兔抗鼠IL-1α多克隆抗体,并对抗体特性进行初步的鉴定。方法:利用RT-PCR技术,从BALB/c小鼠脾脏cDNA中扩增出IL-1α的全长基因,酶切后连接至pET32a(+)原核表达载体,重组载体测序正确后转化至BL21(DE3)大肠杆菌。利用蛋白质原核表达自动诱导方案成功表达重组蛋白。重组蛋白经电洗脱纯化后用以免疫新西兰大白兔,获得了抗小鼠IL-1α的多克隆抗体,ELISA检测抗体效价。Western blot和流式细胞术(FCM)检测抗血清的特异性。结果:成功构建了重组表达载体pET32a(+)-IL-1α,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可达1∶25 600。Western blot和FCM分析该抗体能特异性结合IL-1α。结论:利用重组的IL-1α蛋白成功制备了高效价、高特异性的兔抗IL-1α抗体,为进一步研究IL-1α的生物学功能奠定了基础。
AIM： To construct a recombinant plasmid encoding mouse IL-1α（mIL-1α）,express and purify mIL-1α protein,and prepare its polyclonal antibody.METHODS： The cDNAs were obtained from the spleen cells of BALB/c mice and the full length of mIL-1α gene was amplified by RT-PCR.Then the mIL-1α gene was inserted into a prokaryotic expression vector pET32a（＋） and the resulting recombinant plasmid was transformed into E.coli BL21（DE3）.After auto-induction,the mIL-1α protein was expressed and purified by electro-elution.An anti-mIL-1α polyclonal antibody was raised in New Zealand rabbits after immunization with the purified mIL-1α and the titer was determined by ELISA.The specificity of the polyclonal antibody was identified by Western blot and flow cytometry.RESULTS： The recombinant prokaryotic expression vector pET32a（＋）-IL-1α was successfully constructed,and the mIL-1α protein was expressed and purified.ELISA showed the titer of the anti-mIL-1α serum was 1∶25 600.Western blot and flow cytometry demonstrated the high specificity of the polyclonal antibody to IL-1α.CONCLUSION： The rabbit anti-mIL-1α polyclonal antibody with high titer and specificity has been prepared after immunization with the purified mIL-1α protein,facilitating further functional studies of IL-1α.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第9期989-992,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金重大研究计划培育项目(91029703)
