小鼠B7-H4重组蛋白的表达及鉴定

Expression and identification of recombinant mouse gene B7-H4

目的:构建小鼠B7-H4胞外区基因的真核表达载体,转染酵母细胞以表达mB7-H4,并检测其生物学活性。方法:以mB7-H4的重组质粒为模板,在克隆引物上加入XhoI和EcoRI酶切位点,并用PCR方法扩增得到mB7-H4胞外区的基因片段,经XhoI和EcoRI双酶切后接入酵母表达载体Ppic9上,构建成Ppic9-mB7-H4重组质粒。经双酶切和核酸测序鉴定,将该重组质粒转染至酵母细胞,用PCR、Western Blot及ELISA等方法检测重组基因的表达,并鉴定表达蛋白的生物学活性。结果:转染Ppic9-mB7-H4重组质粒的酵母细胞内表达mB7-H4,诱导表达的mB7-H4能有效抑制CD3单抗活化的T淋巴细胞的增殖(相对抑制率为28.3%)和IL-2(相对抑制率为68.8%)、IL-4(相对抑制率为78.8%)、IL-10(相对抑制率为67.6%)、IFN-γ(相对抑制率为77.7%)等细胞因子的分泌。结论:成功构建了mB7-H4真核表达系统,并获得了有生物学活性的mB7-H4分子。

Objective： To construct a eukaryotic expression vector encoding the gene of extracellular region of mouse B7-H4,to express it in yeast cell line and to determine its biological activity. Methods： The extracellular region of the mouse B7-H4 gene was amplified with Xho I and EcoR I by PCR from a mouse B7-H4 chimeric plasmid. Digested with Xho I and EcoR I,the mB7-H4 gene was inserted into the yeast expression plasmid Ppic9. The DNA sequence was confirmed by double digestion and the Ppicg-mB7-H4 plasmid was transfected into the yeast cells. The expression of mB7-H4 was confirmed by PCR, Western Blot and ELISA analysis, and its biological function was determined. Results： Ppic9-mB7-H4 transfectants expressed mB7-H4 in yeast cells,and mB7-H4 effectively inhibited the proliferation of T lymphocytes with a fractional inhibition rate of 28. 3% and inhibited IL-2, IL 4,IL-10 and IFN-γ production with fractional inhibition rates of 68.8 %, 78.8 %, 67.6 % and 77.7 %, respectively. Conclusion ： The eukaryotic expression plasmid mouse BT-H4 has been successfully constructed and the expressed products of BT-H4 possess biological activity.