【摘要】 目的探讨小鼠B7-H4原核表达载体的构建、表达及多克隆抗体的制备。方法采用特异性引物扩增小鼠B7-H4(mB7-H4)胞外功能区DNA,经酶切、拼接构建原核表达载体pET28a-mB7-H4。将构建的重组表达质粒转化入E.coliBL21(DE3)菌株,采用IPTG诱导表达、Ni-NTA柱亲和层析纯化目的蛋白、SDS-PAGE分析蛋白纯度。将纯化的目的蛋白免疫家兔制备多克隆抗体,并对其进行纯化及鉴定。结果序列测定证实了构建的pET28a-mB7-H4重组表达载体含有mB7-H4编码序列,其序列分析与GenBank中公布序列对比一致,质粒在E.coli中诱导表达相对分子质量(Mr)为26.5 KD的目的蛋白,SDS-PAGE分析表明纯化后的目的蛋白达到电泳纯。双向琼脂扩散法检测抗体效价为1∶16,ELISA法检测抗体效价为1∶12 800,Western blot分析显示抗体能特异性结合mB7-H4。结论成功制备了高表达重组mB7-H4蛋白的原核表达载体及高效价抗mB7-H4多克隆抗体,为进一步研究B7-H4的功能奠定了基础。更多还原

【Abstract】 Objective To study the construction and expression of a prokaryotic expression vector for murine B7-H4 and preparation of a polyclonal antibody.Methods Murine B7-H4(mB7-H4)gene was amplified with specific primers.The PCR product was digested with enzymes and then ligated to reconstruct recombinant prokaryotic expression vector pET28a-mB7-H4 that expresses the mouse B7-H4 and 6 His fusion protein.The expression vector was transformed into E.coli BL21(DE3) and induced with IPTG to express mB7-H4 fusion protein that was then purified with Ni-NTA Purification System and analyzed with SDS-PAGE.The purified fusion protein was used to prepare polyclonal antibody with rabbit.Results The sequence analysis confirmed that this recombinant expression vector contained mB7-H4 coding sequence that was identical with the published sequence in GenBank.The plasmid expressed a 26.5kD protein.After purified with Ni-NTA Purification System,the resulted protein was analytically pure according to the analysis with SDS-PAGE.The titer of the antiserum were 1∶16 and 1∶12800 detected by double diffusion test and ELISA,respectively.Western blot analysis demonstrated that the anti-mB7-H4 antibody bound specifically to mB7-H4.Conclusion The prokaryotic expression vector and the antiserum for mB7-H4 were prepared successfully,which established a foundation for the further study of B7-H4.更多还原