Abstract:

Objective    To construct an eukaryotic expression vector of murine T cell immunoglobulin mucin 3(Tim-3), and express Tim-3 in the melanoma cell line B16. Methods   The murine Tim-3 coding region was amplified by reverse transcription PCR （RT-PCR）, using RNA from spleen cells as a template. The Tim-3 cDNA was cloned into the eukaryotic expression vector pTARGET by T-A cloning. The recombinant vector was transfected into the melanoma cell line B16 by Lipofectamine. Tim-3 expression was analyzed by RT-PCR and Western blot. Results    The murine Tim-3 expression vector was identified by enzyme digestion and sequencing. RT-PCR and Western blot results showed specific Tim-3 expression in B16 cells transfected with the recombinant vector. Conclusion    The Tim-3 coding region was successfully cloned into the eukaryotic expression vector and highly expressed in the melanoma cell line B16.

Key words: Genes; Cloning， molecular； DNA， recombinant； Eukaryotic expression; Mice