Department of Immunology, School of Medicine, Yangzhou University, Yangzhou 225001, PR China.
Plasmid. 2011 May;65(3):239-45. doi: 10.1016/j.plasmid.2011.02.004. Epub 2011 Mar 4.
Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1ε, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1ε gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1ε gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1ε was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1ε in recombinant BaF3/mb15/RAE-1ε cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1ε cells had the ability of promoting NK cells proliferation and IFN-γ secretion. In conclusion, BaF3/mb15/RAE-1ε cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs.
白细胞介素 15（IL-15）是一种关键细胞因子，可促进一类特定免疫细胞的增殖和激活，如自然杀伤（NK）细胞、IFN 产生的杀伤树突状细胞（IKDC）和 CD8 T 细胞。RAE-1ε 是激活型 NKG2D 受体的配体，在 NK 细胞和 IKDC 细胞的增殖和激活中也发挥着重要作用。在本研究中，通过 SOE-PCR 或 PCR 扩增获得了白细胞介素 15（mb15）的膜结合形式编码序列和 RAE-1ε 基因。然后，将扩增的 mb15 和 RAE-1ε 基因分别消化并插入 pVITRO2-mcs 载体的多克隆位点 1（MCS1）和 MCS2 中。成功构建了共表达 mb15 和 RAE-1ε 的重组真核表达载体。将其转染至 BaF3 细胞后，通过 RT-PCR、western blot 和 FCM 分析验证了重组 BaF3/mb15/RAE-1ε 细胞中 IL-15 和 RAE-1ε 的表达。此外，BaF3/mb15/RAE-1ε 细胞具有促进 NK 细胞增殖和 IFN-γ 分泌的能力。总之，成功构建了 BaF3/mb15/RAE-1ε 细胞，这对于进一步的研究非常有用，特别是对于 NK 细胞和 IKDC 等某些免疫细胞亚群的扩增和激活。
