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目的:探讨人FcγRⅠ(huFcγRⅠ)胞外区蛋白的原核表达纯化及其在体外与IgG的结合活性。方法:PCR方法从人的外周血白细胞中扩增出huFcγRⅠORF区全长基因并与T载体链接,从克隆载体huFcγRⅠ-T中扩增huFcγRⅠ胞外区基因,并将其装入原核表达载体pGEX-6p-1。构建好的载体转化大肠杆菌BL21(DE3),以IPTG诱导重组蛋白表达,并利用SDS-PAGE和Western blot对表达的蛋白进行鉴定。结果:扩增到了huFcγRⅠ的胞外区基因,所构建的pGEXhuFcγRⅠ重组质粒经PCR和酶切鉴定与预期一致。IPTG诱导下目的蛋白的表达率约为30%。SDS-PAGE结果显示表达的目的相对分子质量(Mr)56 700与理论预期值一致。West-ern blot结果显示原核表达的huFcγRⅠ胞外区蛋白与人IgG特异亲和。结论:FcγRⅠ(huFcγRⅠ)胞外区蛋白在原核表达系统中得到有效的表达,复性蛋白在体外与人的IgG特异结合。
Objective: To investigate the prokaryotic expression and purification of extracellular domain of human FcγRⅠ (huFcγRⅠ) and its binding activity to IgG in vitro. Methods: The full-length huFcγRⅠORF gene was amplified from human peripheral blood leucocytes by PCR and linked to the T vector. The huFcγRⅠ extracellular region gene was amplified from the cloning vector huFcγRⅠ-T and inserted into the prokaryotic expression vector pGEX- 6p-1. The constructed vector was transformed into E. coli BL21 (DE3) to induce recombinant protein expression with IPTG, and the expressed protein was identified by SDS-PAGE and Western blot. Results: The extracellular region of huFcγRⅠ was amplified. The constructed recombinant plasmid pGEXhuFcγRⅠ was identified by PCR and restriction enzyme digestion. IPTG induced protein expression rate of about 30%. The result of SDS-PAGE showed that the relative molecular mass of Mr 56 700 was consistent with the expected value. West-ern blot results showed that the prokaryotic expression of huFcγRI extracellular region protein and human IgG specific affinity. CONCLUSION: The extracellular domain of FcγRⅠ (huFcγRⅠ) is efficiently expressed in prokaryotic expression system. Refolded protein specifically binds human IgG in vitro.