关键词: 巨噬细胞, 小鼠, Tim-4, 基因

Abstract: To establish a new macrophage cell line of murine Tim-4. MethodsPeritoneal 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. ResultsProducts of RT-PCR amplification were digested with BamHⅠand HindⅢ，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. ConclusionAn 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.

Key words: Gene, Tim-4, Macrophage, Mouse