小鼠FasL全长cDNA的克隆、真核表达载体构建及其在树突状细胞中的表达

Cloning and eukaryotic expression vector construction of mouse FasL gene and its expression in dendritic cells

目的克隆小鼠FasL(mouse FasL,mFasL)全长cDNA,构建其真核表达载体,并验证其在小鼠骨髓来源的树突状细胞(dendritic cells,DC)中的表达。方法采用RT-PCR和TA克隆技术,从激活的小鼠脾脏T淋巴细胞中扩增mFasL全长cDNA,亚克隆到T载体中,再克隆到pIRES2EGFP载体中。转染小鼠DC后,用RT-PCR、免疫荧光及W estern blot检测mFasL mRNA和蛋白表达。结果测序证实所得的mFasL cDNA序列与其在GenBank中的序列完全一致。mFasL真核表达载体转染小鼠DC后,能表达mFasL mRNA和蛋白。结论成功克隆了mFasL基因,构建其真核表达载体,并证明能有效表达于DC中。

Objective To clone mouse FasL （mFasL） full length cDNA and construct eukaryotic expression vector, and to detect its expression in dendritic cells. Methods RT-PCR and TA cloning technique were used to clone the mFasL full length cDNA from mouse activated T lymphocytes, and then eukaryotic expression vector plRES2EGFP containing mFasL cDNA was constructed. The positive recombinants plRES2EGFP-mFasL were transfected into dendritic cells （DC） , the expression of mFasL mRNA was detected by RT-PCR and the expression of mFasL protein was assayed by immunofluorescence and Western blotting. Results The cloned full reading frame of mFasL cDNA was in coincidence with the sequence registered in GenBank. In the DC after transfection of plRES2EGFP-mFasL, the expressions of mFasL mRNA and mFasL protein were detected. Conclusion The mFasL cDNA was cloned and constructed into the eukaryotic expression vector successfully, and mFasL gene could be expressed efficiently in DC transfected by plRES2EGFP-mFasL.