Construction of eukaryotic expression vector for human T cell immunoglobulin mucin protein-4 and its expression in 16HBE cells

Abstract：In this study, we construct a eukaryotic expression vector of the entirely coding human T cells immunoglobulin mucin protein-4 (TIM-4) and transfect it into 16HBE cell line, for benefiting the further study on biological function of TIM-4. According to TIM-4 cDNA sequence in GeneBank (NO :NM-138379.2), we designed two primers which have sites of EcoRI and BamHI, respectively. The TIM-4 cDNA was amplified by RT-PCR from human bone marrow and then cloned into pMD18-T vector to obtain recombinant vector pMD18-T-TIM-4, which was verified by colony PCR, double enzyme digestion, and sequencing. Then the identified TIM-4 cDNA fragment was subcloned into pEGFP-C2 plasmid. The recombination vector pEGFP-C2-TIM-4 was identified by colony PCR,double enzyme digestion and sequencing. After confirmation with sequence analysis, the expression vector pEGFP-C2-TIM-4 was transfected into the 16HBE cells, and then the expression of TIM-4 in transfected cells was detected by Real-Time PCR. The entirely coding human TIM-4 gene fragment was about 1 134 bp. The recombinant pEGFP-C2-TIM-4 was identified by colony PCR, and the amplification fragment was about 1 134 bp. By double enzyme digestion, the recombinant was digested into about 1134 bp and 4.7 kb fragments. The DNA sequencing revealed the insert ed fragment was identical with human TIM-4 cDNA in GeneBank. Green fluorescence could be observed in the pEGFP-C2-TIM-4-transfected cells; the expression of TIM-4 was significantly increased in the transfected cells. In our studies, the human TIM-4 cDNA was amplified from human bone marrow for the first time; furthermore its eukaryotic expression vector pEGFP-C2-TIM-4 was successfully constructed and highly expressed in 16HBE cells, which will provide a foundation for the further research.