摘要
目的建立研究小鼠Tim-4功能的细胞模型。方法常规分离小鼠腹腔巨噬细胞,RT-PCR扩增Tim-4全长基因片段,构建含Tim-4全基因片段的真核表达载体pcDNA3.0-Tim-4,通过BamHⅠ、HindⅢ双酶切及测序鉴定其正确性。利用脂质体将pcDNA3.0和pcDNA3.0-Tim-4分别转染小鼠巨噬细胞系RAW264.7,通过G418加压筛选,建立稳定高表达Tim-4的RAW264.7,并采用RT-PCR、流式细胞术和免疫细胞化学染色法检测Tim-4 mRNA和蛋白的表达水平。结果RT-PCR扩增产物经BamHⅠ、HindⅢ双酶切,与pcDNA3.0连接构建的重组体经酶切和测序,结果与GenBank报道序列一致。RT-PCR结果显示,RAW264.7-T4细胞表达Tim-4 mRNA的水平显著高于对照组,流式细胞术显示RAW264.7-T4高水平表达Tim-4蛋白,免疫细胞化学染色表明Tim-4主要表达于胞膜。结论成功构建了携载小鼠Tim-4全基因的表达载体,并用该重组体建立了稳定高表达Tim-4 mRNA和蛋白的小鼠巨噬细胞系,为进一步探讨Tim-1与Tim-4的相互作用及对免疫细胞功能的影响奠定基础。
Objective To establish a new macrophage cell line of murine Tim-4. Methods Peritoneal macrophages were regularly separated from mice. RT-PCR was used to amplify the full gene fragment of murine Tim-4, with which to construct the pcDNA3.0-Tim-4 expression vector. Restriction endonucluease digestion and sequencing were used to verify its correctness. Also, RAW264.7 cells were separately tansfected with pcDNA3.0 and pcDNA3 .0-Tim-4 by liposomes. After screening with a high level of G418, a new cell line expressing stable and high Tim-4 was established. The mRNA and protein levels were further determined by RT-PCR, FCM and immunochemistry staining. Results Products of RT-PCR amplification were digested with BamH 1and Hindm, then Tim-4 was cloned into pcDNA3.0 and pcDNA3.0-Tim-4 recombinant was constructed. The result was consistent with the sequence of GenBank after digestion and sequencing. The mRNA level of RAW264.7-Tim-4 was significantly higher than that of the control group, as were proteins expressed in these cells, which was shown by FCM and immunochemistry staining. Also, Tim-4 expression was mainly found on the surface of the macrophage and cytoplasmic expression was also found. Conclusion An expression vector with the full murine Tim-4 gene and a new line with stable and high expression of Tim-4 were successfully established, which will set a good basis for further study on the interaction of Tim- 1 and Tim-4 and its impact on immunocytes.
出处
《山东大学学报（医学版）》
CAS
北大核心
2008年第6期551-555,共5页
Journal of Shandong University:Health Sciences
基金
山东省优秀中青年科学家科研奖励基金资助课题(2004BS02018)
山东省卫生厅青年基金资助课题(JZ13)
