H-2K^d基因siRNA表达质粒的构建及在小鼠LAK细胞的表达

Construction of a plasmid expressing siRNA aimed at the H-2K^d gene and expression of H-2Kd in mouse LAK cells

目的构建针对BALB/C小鼠H-2Kd基因siRNA表达质粒,观察其在小鼠LAK细胞的表达,为进一步研究H-2Kd基因功能奠定基础。方法设计siRNA干涉靶序列,体外合成两段互补的寡核苷酸,通过与线性化的pSi-lencer 3.0-H1连接,转化大肠杆菌DH5a扩增纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列。采用siRNA表达质粒封闭小鼠LAK细胞MHC-I(H-2Kd)的表达,同时设空转染组和无关序列组,流式细胞术检测不同组别靶蛋白的表达。结果纯化的质粒分子量为2.8 Kb,插入的寡核苷酸序列与设计的序列完全相符,流式细胞术检测证实siRNA能够抑制靶蛋白的表达。结论成功构建了针对H-2Kd基因的siRNA表达质粒并抑制了其表达。

Objective To construct the siRNAs expressing plasmid aiming at the H-2K^d gene and to explore the H-2K^d expression in mouse LAK cells and function of H-2K^d with the RNA interference technology. Methods First, a target sequence of the H-2K^d gene was selected, then according to the sequence, two complementary oligonucleotides were lisated into psilencer 3.0- H1, which was transformed into DH5a bacteria, and then it was amplified and purified to get the plasmid. The purified plasmid was identified by gel electrophoresis and sequencing. H-2K^d-targeting siRNA plasmid was used in spleen lymphocytes of BALB/C mice. Flow cytometry（FCM） was used to determine the expression of H-2K^d on the transfected and control cells. Resets Gel electrophoresis and sequencing results showed that the plasmid was about 2.8 kb which was identical with the positive control, and the sequence was identical with what had been inserted. Also siRNA treatment significantly inhibited the expression of the targeted protein. Conclusion siRNAs expression plasmids aiming at the H-2K^d gene have been successfully constructed and could inhibit the H-2K^d expression.