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目的:表达并纯化重组人BTLA(hBTLA)分子胞外区,制备和鉴定兔抗hBTLA多克隆抗体,为进一步实验研究奠定基础。方法:采用特异性引物扩增hBTLA胞外功能区DNA,经酶切、连接构建原核表达载体pET28a-hBTLA。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电泳分析蛋白纯度。将纯化的目的蛋白免疫家兔制备多克隆抗体,并对抗体进行纯化、效价测定及特异性鉴定。结果:序列测定证实构建的pET28a-hBTLA重组表达载体含有hBTLA编码序列,其序列比对分析与GenBank中公布序列一致,质粒在E.coli中诱导表达相对分子质量(Mr)为15720的目的蛋白,SDS-PAGE电泳分析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∶20 000,Western blot分析显示抗体能特异性结合重组hBTLA蛋白。结论:成功构建了hBTLA蛋白的原核表达载体,获得高纯度的融合蛋白,制备高效价、高特异性的多克隆抗体。
OBJECTIVE: To express and purify the extracellular domain of recombinant human BTLA (hBTLA), and to prepare and identify the polyclonal anti-hBTLA antibody, which will lay the foundation for further experimental research. Methods: DNA of hBTLA extracellular domain was amplified by specific primers. The prokaryotic expression vector pET28a-hBTLA was constructed by restriction enzyme digestion. The constructed recombinant plasmid was transformed into E. coli BL21 (DE3) strain and expressed by IPTG. Purification of the target protein by Ni-NTA affinity chromatography and protein purity by SDS-PAGE electrophoresis. The purified target protein was immunized rabbit to prepare polyclonal antibody, and the antibody was purified, titer and specific identification. Results: Sequence analysis confirmed that the constructed recombinant plasmid pET28a-hBTLA contained the hBTLA coding sequence. The sequence alignment analysis was consistent with the published sequence in GenBank. The target protein was expressed in E.coli with the relative molecular mass (Mr) of 15720, Purified SDS-PAGE electrophoresis analysis of the target protein reached electrophoretic purity. The titer of the antibody was 1:16 by two-dimensional agar diffusion assay, and the titer of the antibody was 1:20 000 by ELISA. Western blot analysis showed that the antibody could specifically bind to the recombinant hBTLA protein. CONCLUSION: The prokaryotic expression vector of hBTLA protein was successfully constructed and the fusion protein of high purity was obtained. The polyclonal antibody with high titer and high specificity was prepared.