小鼠B7-H4基因的真核表达及其对淋巴细胞增殖的影响

目的构建小鼠B7-H4胞外段的真核表达载体,观察其在体外对淋巴细胞增殖的影响,为深入研究B7-H4在T细胞活化及移植排斥反应中的作用提供实验材料。方法提取小鼠肺、脾脏总RNA,RT-PCR反转录cDNA,以此为模板,扩增B7-H4胞外段基因,将其导入pGEM-T Easy载体,构建TA-mB7-H4质粒。用XBaI和HindIII双酶切后琼脂糖凝胶电泳分析和测序鉴定。将测序证实的mB7-H4酶切后装入MYC-HIS-EGFP-N荧光表达载体中,构建B7-H4-EGFP真核表达载体,转化JM109感受态细菌,提取重组质粒,酶切后琼脂糖凝胶电泳分析和测序鉴定。同时构建control-EGFP载体。应用脂质体法将重组质粒转染CHO细胞,经G418筛选,获得稳定表达B7-H4-EGFP的CHO细胞株,用MTT分析其分别对BALB/c小鼠、C57小鼠淋巴细胞和二者混合淋巴细胞增殖的影响。结果经测序证实,所克隆的小鼠B7-H4 cDNA和构建的重组质粒基因序列正确;转染的CHO细胞能稳定地表达跨膜型重组蛋白B7-H4;表达的B7-H4对淋巴细胞增殖具有明显抑制作用。结论成功构建了B7-H4真核表达系统,能表达有生物学活性的B7-H4分子,为进一步探讨B7-H4在T细胞活化和移植排斥反应中的作用奠定了基础。

Expression of B7-H4 gene from mouse in eukaryofic system and its suppressive effect on proliferation of lymphocytes cell

Objective To provide subjects for lucubrating the effect of B7-H4 on T cell activation and graft rejective reaction through cloning and constructing the eukaryotic expression vector encoding the gene of extracellular region of B7-H4 from mouse,and investigate the effect of B7-H4 on the proliferation of lymphocytes in vitro.Method The total RNAs of mouse lung and spleen were extracted and cDNA was transcribed from RNA using RT-PCR technique.The gene of extracellular region of B7-H4 was amplified according to the template of cDNA by PCR.The amplified cDNA was imported into pGEM-T Easy vector to construct TA-mB7-H4 plasmid.The plasmid was cut by restriction enzyme of XBaI and HindIII and was identified by the agarose gel electrophoresis and sequence scanning.Then the mB7-H4 corroborated by sequencing was inserted into the fluorescence expression vector MYC-HIS-EGFP-N after cut by the restriction enzymes to construct B7-H4-EGFP and control-EGFP eukaryotic expression vectors.The recombinant plasmids were transfected into JM109 competence bacteria,and were extracted and identified by the agarose gel electrophoresis and the sequencing after cut with the restriction enzymes.They were transfected into CHO cell through lipofectamineTM 2000,and the CHO cell lines expressing stably the fusion protein were obtained through G418 selection.MTT colorimetry was used to assess the effect of B7-H4 on the proliferation of lymphocyte in the culture of lymphocyte from BALB/c or C57 mouse respectively and in co-culture of lymphocyte from both BALB/c and C57 mouse.Result The gene sequences of B7-H4 cDNA cloned from mouse and TA-mB7-H4 constructed were correct by sequencing.The transfective CHO cells stably expressed the recombinant transmembrane B7-H4 protein.The B7-H4 protein suppressed the lymphocyte proliferation either in the culture of lymphocyte respectively and in co-culture of lymphocyte from BALB/c and C57 mouse.Conclusion The B7-H4 eukaryotic expression vector was constructed successfully and can suppress recombinant B7-H4 protein with biological activity.It lay the foundation for further studies on the role of B7-H4 in transplant rejection and T cell activation.