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应用RT PCR方法 ,从PHA活化的扁桃体T细胞的mRNA中 ,扩增获得编码人OX4 0分子的cDNA ,并将其克隆到pMD18 T载体 ,经PCR、酶切和测序鉴定确证。进而构建pcDNA3 1 OX4 0重组真核表达载体 ,脂质体法转染L92 9细胞 ,经G4 18筛选 ,获得能稳定高表达OX4 0分子的细胞株。经OX4 0 FITC单抗标记和FCM检测 ,阳性表达率为 89 1%。转基因细胞在可溶性CD4 0L共存条件下 ,与未成熟DC共育 ,能促进DC的进一步分化成熟 ,使DC膜分子CD4 0、CD86和CD83呈上调性表达。
The RT-PCR method was used to amplify cDNA encoding human OX40 from PHA-activated Tonsil T cells. The cDNA encoding human OX40 was cloned into pMD18 T vector and confirmed by PCR, restriction enzyme digestion and sequencing. Furthermore, a recombinant eukaryotic expression vector pcDNA3 1 OX4 0 was constructed and transfected into L92 9 cells by lipofectamine. The cell lines stably overexpressing OX4 0 were obtained by G418 selection. OX4 0 FITC monoclonal antibody labeling and FCM detection, the positive rate of 89 1%. The co-culture of transgenic cells with immature DC under the coexistence of soluble CD40L can promote the further differentiation and maturation of DC, and upregulate the expressions of CD4 0, CD86 and CD83.