小鼠FasL基因的克隆和在软骨细胞中表达

The cloning of mouse FasL gene and expression in chondrocytes

目的 :获得小鼠FasL(mFasL)的cDNA克隆 ,通过逆转录病毒表达系统 pLNCX2 在软骨细胞中进行表达 ,并分析其功能。方法 :应用RT PCR和T A克隆技术 ,从激活的小鼠脾脏淋巴细胞总RNA中 ,扩增mFasLcDNA并克隆到T载体中 ,再亚克隆到 pLNCX2 中。转染软骨细胞后 ,以流式细胞仪检测mFasL蛋白的表达 ,以单向混合淋巴细胞反应鉴定其活性。结果 :成功地克隆了 0 .85 5kb的mFasLcDNA ,并经测序确证。通过 pLNCX2 转染软骨细胞 ,转染率为 6 0 .6 4 %。流式细胞仪检测到FasL蛋白的表达 ,并能显著抑制同种异体的单向混合淋巴细胞反应 ,刺激指数下调到未转染软骨细胞的11.71%。结论 :克隆到的mFasL基因 ,并能通过 pLNCX2 有效表达。

AIM: To clone mFasL cDNA, then insert it into retrovirus expression system pLNCX 2, and express mFasL cDNA in chondrocytes to study its function. METHODS: RT PCR was applied to amplify mFasL cDNA from the total RNA of activated mouse spleen lymphocytes. The cDNA was inserted into a T cloning vector, then subcloned into pLNCX 2. After transfection of chondrocytes, expression of FasL cDNA was detected by FACS, and activity of expressed product was analyzed by single mixed lymphocyte reaction(SMLC). RESULTS: mFasL cDNA fragment of 0.855 kb was successfully cloned and verifiedby sequencing. The transfection efficiency of mFasL cDNA in chondrocytes determined by FACS was 60.64%. Expressed product could evidently inhibit SMLC of allogeneic lymphocytes. It’s stimulating index (SI) reduced to 11.71% as compare with the untransfected chondrocytes. CONCLUSION: Cloned recombinant pLNCX 2 FasL can effectively express mFasL in chondrocytes, and can evidently inhibit SMLC of allogeneic lymphocytes.