转录因子Blimp-1的原核表达、纯化及抗体制备  

Prokaryotic expression,purification and polyclonal antibody preparation of Blimp-1

作　　者：邓少丽[1] 胡福泉[1] 蹇锐[1] 饶贤才[1] 蒋静[1] 程小星[1] 

机构地区：[1]第三军医大学基础医学部微生物学教研室重庆市微生物工程实验室,重庆400038

出　　处：《第三军医大学学报》2006年第3期205-207,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30370603)~~

摘　　要：目的获得B limp-1纯化蛋白,制备多克隆抗体,为研究浆细胞发育的调控奠定基础。方法采用PCR方法从B limp-1质粒中克隆B limp-1编码前350个氨基酸的基因片段,构建B limp-1与6个H is的融合蛋白原核表达质粒,进行原核表达与蛋白纯化后,免疫动物,制备B limp-1多抗。采用ELISA方法检测其效价,W estern检验抗体特异性及在骨髓瘤细胞株中的表达。结果构建了表达B limp-1前350个氨基酸的原核表达质粒PQE-B limp-1,经过大肠杆菌表达、镍亲和层析柱纯化,得到分子量约46×103的融合蛋白,免疫家兔后得到多抗血清。ELISA显示抗体效价达1/20 000。W esternb lot结果显示此多克隆抗体与B limp-1蛋白特异性结合,并在骨髓瘤细胞中表达。结论本研究获得B limp-1纯化蛋白,制备了B limp-1多克隆抗体,为进一步研究B limp-1的作用机制及与血液疾病间的关系奠定了基础。Objective To obtain purified Blimp-1 protein and prepare anti-Blimp-1 polyclonal antibody. Methods The gene segment consisting of first 350 amino acids of the Blimp-1 ORF was amplified from Blimp-1 plasmid. The prokaryotie expression plasmid containing 6 His was constructed. The plasmid was expressed in E coli. JM109, and the expression product was purified. Rabbits were immunized with the purified Blimp-1 protein and the antiserum was obtained. The titers and specificity of the antibodies were measured by ELISA and Western blotting. Results The plasmid PQE-Blimp-1 was constructed and a molecular weight of 46 103 fusion protein was obtained after induction with IPTG. The anti-Blimp-1 antibody was obtained and purified. The results of ELISA and Western blotting indicated that the polyelonal antibody had high titer and specificity. Conclusion The purified Blimp-1 protein and anti-Blimp-1 polyelonal antibody have been acquired, which lays the foundation for further research on Blimp-1 mechanism in hemopathy.

关 键 词：BLIMP-1 原核表达 抗体制备 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]