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目的:构建s TACI-Fc-Myc重组质粒,并进行原核表达和纯化具有生物活性的融合蛋白。方法:通过PCR法获得s TACI-Fc-Myc重组片段,然后把融合基因片段与原核载体p ET28a连接在一起,并构建p ET28a-s TACI-Fc-Myc重组子,并转入BL21(DE3)中进行表达,用蛋白A凝胶亲和层析柱进行纯化及酶联免疫吸附剂(ELISA)法测定其生物学活性。结果:获得了s TACI-Fc-Myc重组质粒,且该质粒可以在BL21(DE3)中表达,亲和层析柱纯化后纯度可达到95%以上,与BAFF的结合活性具有剂量依赖性,浓度达到5 ng/μL时,两者的吸附达到饱和。结论:成功构建了s TACI-Fc-Myc原核表达载体,并使有生物学活性的融合蛋白在BL21(DE3)上获得了稳定表达,为进一步研究并筛选高活性BAFF拮抗肽奠定了基础。
Objective: To construct s TACI-Fc-Myc recombinant plasmid, and to prokaryotic express and purify the bioactive fusion protein. METHODS: The s TACI-Fc-Myc recombinant fragment was obtained by PCR. The fusion gene fragment was then ligated with the prokaryotic vector p ET28a to construct pET28a-s TACI-Fc-Myc recombinant. The recombinant plasmid was transformed into BL21 (DE3) , And purified by Protein A gel affinity chromatography and its biological activity was determined by enzyme-linked immunosorbent assay (ELISA). Results: The recombinant plasmid of TACI-Fc-Myc was obtained and the plasmid was expressed in BL21 (DE3). Purity of the recombinant plasmid was over 95% after purification. The binding activity to BAFF was dose- At 5 ng / μL, the adsorption of both reaches saturation. CONCLUSION: The prokaryotic expression vector of s TACI-Fc-Myc was successfully constructed and the biologically active fusion protein was stably expressed on BL21 (DE3), which laid the foundation for further study and screening of highly active BAFF antagonist.