人B7-H4基因转染细胞株的构建及其对T细胞的作用

目的克隆人B7-H4基因,构建人B7-H4基因转染细胞株并初步研究其对T细胞的作用。方法通过RT-PCR的方法获得人B7-H4分子的基因,将目的片段双酶切(EcoRⅠ和BamHⅠ)后与pIRES2-EGFP真核表达载体连接,构建重组真核表达载体pIRES2-EGFP-B7-H4并进行鉴定;脂质体转染法将重组载体导入L929细胞,经G418加压筛选并通过流式细胞术和RT-PCR检测表达载体是否转入L929细胞中,通过检测活化T细胞上CD25,CD69的表达及T细胞增殖实验对B7-H4转基因细胞的生物学功能进行初步研究。结果从外周血活化单核细胞中扩增出目的基因,构建重组表达载体pIRES2-EGFP-B7-H4并将其转染至L929细胞,构建了一株稳定表达人B7-H4分子的基因转染细胞株,对其生物学功能的研究显示B7-H4分子可以下调活化T细胞上CD25,CD69的表达,并且对T细胞的活化增殖起到抑制作用(P<0.05)。结论成功构建了稳定表达B7-H4分子的基因转染细胞株,为进一步研制B7-H4分子的单克隆抗体提供了有效的免疫原。

Establishment of human B7-H4 transfected cell line and its effects on T cell

Objective To clone the human B7-H4 gene and construct the human B7-H4 transfected cell line,and to explore its effects on T cell. Methods Human B7-H4 gene was amplified by RT-PCR.Target fragment was digested with restriction endonucleases EcoRⅠ and BamHⅠ,and then was inserted into eukaryotic expression vector pIRES2-EGFP to construct the recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4.The recombinant plasmid was transfected into L929 cell line with LipofectamineTM 2000,and the transfected cells were further screened with G418.Flow cytometry and RT-PCR were used to verify whether the expression vector was inserted into L929 cell line.The biological functions of transfected cells were researched with T cell proliferation test by detecting the expression of CD69,CD25 on activated T cells. Results The target gene was successfully amplified from activated monocytes,and a stable cell line expressing human B7-H4 was successfully established after recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4 was constructed and transfected into L929 cells.The results of biological function showed that B7-H4 down-regulated the expression of CD25,CD69 on activated T cells and inhibited T cell proliferation(P<0.05). Conclusion The transgenic cell line stably expressing human-B7-H4 molecule is successfully obtained,which provide an effective immunogen for preparing anti-B7-H4 monoclonal antibody.