小鼠CD_(40L)基因的克隆与真核细胞表达研究

Cloning and Expressing Mouse CD_(40) Ligand

目的构建含有小鼠CD40L基因的重组真核表达载体,并鉴定其在转染细胞中的表达。方法自活化的T细胞经RT-PCR得到小鼠CD40L的cDNA基因,将其克隆入真核表达载体pCMV-Myc,获得重组质粒pCMV-Myc/CD40L,酶切及测序鉴定;脂质体法转染CHO细胞,使用RT-PCR及流式细胞技术分别从mRNA和蛋白水平对CD40L的表达进行检测。结果将pCMV-Myc/CD40L真核表达载体转染CHO细胞,RT-PCR和流式细胞仪检测证实小鼠CD40L在真核细胞中暂态表达。结论成功地建立表达小鼠CD40L基因的重组真核表达载体,为进一步研究其生物学特性及肿瘤的基因治疗提供了基础。

Objective To construct a recombinant eukaryotic vector expressing mCD40L gene and to identify the expression product in CHO cells. Methods The total RNA from activated T cells were isolated at first, reversed transcript reaction and PCR were then used by Oligod （T） n primer and mCD40L pair primers respectively, cDNA sequences were cloned into vector expressing pCMV-Myc followed by digestion and sequencing to identify the recombinant eukaryotic vector. The pCMV-Myc/CD40L was transfected into CHO cells by lipidosome methods, and the expressed mCD40L was identified by RT-PCR and flow cytometry. Results mCD40L gene was confirmed to be correctly inserted into eukaryotic vector pCMV-Myc based on the evidences of endonulease digestion and sequencing, which was observed in the transfected CHO cells with RT-PCR and flow cytometry. Conclusion The recombinant eukaryotic vector expressing mCD40L gene has been successfully constructed, which can be used for further research in tumor gene therapy.