转录因子Blimp-1的原核表达、纯化及抗体制备

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。

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

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 prokaryotic 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 polyclonal antibody had high titer and specificity.Conclusion The purified Blimp-1 protein and anti-Blimp-1 polyclonal antibody have been acquired,which lays the foundation for further research on Blimp-1 mechanism in hemopathy.