人B7-H1-Fc融合分子的构建及其在CHO细胞中的表达与鉴定

Construction,expression and identification of human B7-H1-Fc chimeric molecule in mammalian CHO cell

目的利用基因工程手段构建hB7-H1-Fc重组cDNA,用真核表达系统制备有活性的hB7-H1-Fc融合蛋白。方法PCR方法从活化的人T细胞cDNA文库中扩增编码人B7-H1膜外区的cDNA序列,将其与人IgG1Fc和pC I-neo片段连接,构建成hB7-H1-Fc重组表达载体。用脂质体法转染CHO细胞,夹心ELISA法检测上清中融合蛋白hB7-H1-Fc的表达,经Prote in A亲合层析纯化,SDS-PAGE、免疫印迹鉴定表达产物。结果PCR扩增得到编码人B7-H1膜外区的727bp基因片段,与hIgG1Fc的cDNA片段一起连接并插入pC I-neo表达质粒。重组质粒转染CHO细胞后,夹心ELISA法检测显示培养上清中有hB7-H1-Fc蛋白表达。纯化后的hB7-H1-Fc蛋白经SDS-PAGE和免疫印迹鉴定其分子质量约51.76×103,与理论预测值相符。结论成功构建了hB7-H1-Fc重组表达载体,利用哺乳动物细胞CHO表达出有活性的hB7-H1-Fc融合蛋白,为进一步研究B7-H1分子在免疫耐受、自身免疫性疾病中的作用奠定了基础。

Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region- hIgGlFc-pCI-neo eukaryotic expression vector, and express the functional fusion protein in mammalian CHO cell. Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR, fused with hIgGlFc, then transformed into pCI-neo expression vector and verified by sequencing. The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent. The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein, and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography. The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1. Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgGlFc into the eukaryotic expression vector pCI-neo. After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent, the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA. The immune activity of the fusion protein was verified by Western blotting, and its molecular weight was about 51. 76×10^3 , very close to the expected value. Conclusion The hB7 -H1 -Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance, autoimmune diseases.