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目的:制备纯化重组蛋白mFcγRⅢ,并鉴定其生物学活性。方法:从克隆载体mRⅢ-T中扩增mFcγRⅢ胞外区,构建原核表达载体pETmRⅢ,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,SDS-PAGE和Westernblot对重组表达蛋白进行鉴定;表达产物通过包涵体纯化后用稀释方法复性;ELISA检测复性蛋白活性。结果:成功构建了重组表达质粒pETmRⅢ,纯化和复性了重组蛋白mFcγRⅢ;复性的重组蛋白mFcγRⅢ具有较强的体外活性。结论:原核重组蛋白mFcγRⅢ复性后具有较强的体外结合配体特性,为探索重组蛋白治疗自身免疫病奠定了基础。
Objective: To prepare purified recombinant protein mFcγRⅢ and identify its biological activity. Methods: The mFcγRⅢ extracellular domain was amplified from the cloning vector mRⅢ-T. The prokaryotic expression vector pETmRⅢ was constructed and transformed into E. coli BL21 (DE3). The recombinant protein was induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blot. Refolded by inclusion body purification and dilution method; ELISA test for refolding protein activity. Results: The recombinant plasmid pETmRⅢ was successfully constructed and the recombinant protein mFcγRⅢ was purified and renatured. The renaturation of mFcγRⅢ was more active in vitro. CONCLUSION: The prokaryotic recombinant protein mFcγRⅢ has strong binding ligand characteristics in vitro, which lays the foundation for exploring the recombinant protein for the treatment of autoimmune diseases.