小鼠B7H4原核表达载体的构建、表达及多克隆抗体的制备_临床医学论文.docVIP

小鼠B7原核表达载体的构建、表达及多克隆抗体的制备_临床医学论文 小鼠B7原核表达载体的构建、表达及多克隆抗体的制备_临床医学论文 作者：肖德乾，肖欢，胡国艳，那志萍，郑淑华，刘伟，张良清，徐军发 【摘要】 目的 探讨小鼠B7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（mB7）胞外功能区DNA，经酶切、拼接构建原核表达载体pET28a。将构建的重组表达质粒转化入E.coli BL21 (DE3)菌株，采用IPTG诱导表达、Ni柱亲和层析纯化目的蛋白、SDS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重组表达载体含有mB7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。结论 成功制备了高表达重组mB7蛋白的原核表达载体及高效价抗mB7多克隆抗体，为进一步研究B7的功能奠定了基础。 【关键词】 B7；原核表达载体；基因表达；多克隆抗体 　　Abstract: Objective To study the construction and expression of a prokaryotic expression vector for murine B7gene 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. 　　Key words: B7ression; polyclonal antiserum 　　B7（B7x，B7S1）是T细胞活化的共抑制分子（co），其mRNA广泛表达于淋巴样和非淋巴样组织（如肺、睾丸、胰腺、前