sTACI-Fc-Myc重组质粒的构建、原核表达及活性鉴定

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。

Construction, Expression and Activity Analysis of sTACI-Fc-Myc Protein

Objective:To construct recombinant plasmid of sTACI-Fc-myc gene and to induce its prokaryotic express and to purify the protein.Methods:sTACI-Fc-myc gene was obtained by PCR amplification and then combined with pET28a to construct pET28a-sTACI-Fc-myc recombinant plasmid. The recombinant plamid was transformed into BL21 (DE3), and then sTACI-Fc-myc protein was expressed and purified by protein A affinity column chromatography. The purity and activity of sTACI-Fc-myc were identified by ELISA, SDS-PAGE, respectively.Results:The sTACI-Fc-myc gene was obtained and the recombinant plasmid can express in BL21 (DE3). The purity can be as high as 95% after affinity chromatography. The biological activity of the purified sTACI-Fc-myc protein was analyzed. sTACI-Fc-myc could bind BAFF specifically in a dose-dependent manner, and the saturated concentration was 5 ng/uL.Conclusion:The fusion vector is constructed successfully and a purified recombinated sTACI-Fc-myc protein is obtained, which lays a foundation for further studies of BAFF antagonist peptibody.