Cloning Of Human TIM-3 CDNA And Construction Of Eukaryotic Expression

Background and objective:CD4+ effector T cell divide into Th1 and Th2 two subsets according to their cytokine produciton. Th1 and Th2 play a different role in the host immunity. Th1 cell mainly involved in the cellular immune response which was defense agaginst bacteria and virus. Th2 cell mainly involved in the humoral immune response and allergic reaction, etc. Tim (T-cell immunoglobulin and mucin-domain-containing molecule) family members are newly found type I membrane glyeoprotein expressed on T cells. It has a role in the regulation of Th1 and Th2 differentiation and T cell mediated immune responses. Tim-3 is the important one in the Tim family members. It is specifically expressed on Th1 cells, not on naive T cell, B cell. It binds with its ligand galectin-9 and negatively regulates Th1 response. So it could regulate the immune system through the negative regulation of Th1 response. But in recent years, it had found that aside from T cell, Tim-3 is also found expressed on the Th17 cell, Treg cell, macrophage, NK cell, DC cell, mast cell and some kinds of tumor cells. Tim-3 plays a different role in these cells. It is still not clear that its exact function of expression on these cells. These all demonstrate that Tim-3 has other functions which we do not know, so we use the method of RT-PCR to clone the human Tim-3 gene from the PBMC of the human peripheral blood and constructed the recombinant eukaryotic expressing plasmid containing Tim-3 gene and observe its expression on the hepatocellular carcinoma cell and macrophage. This provided a basis for the follow-up function studies of Tim-3 and eventually apply to the clinical gene therapy.Methods:1. Human Tim-3 cDNA was amplified by reverse transcription-polymerase chain reaction(RT-PCR) from total RNA isolated from the PBMC of the health person.2. The PCR product was cloned into pGEM-T vector, which was transformed into Escherichia coli DH5αby using TSS method. Subsequently, the T-A colonies were selected by ampicillin and X-gal, and then confirmed by the analysis of dual- endonuclease digestion and sequencing. 3. The pGEM-T-Tim-3 recombinant plasmid was purified and digested with Bglâ…¡and Salâ… ,then the Tim-3 was inserted into the Bglâ…¡and Salâ… sites of the eukaryotic expression plasmid pEGFP-N1, which contains EGFP report gene and kanamycin resistance gene. The recombinant pEGFP-N1-Tim-3 was transformed into competent cell Escherichia coli DH5αfor propagation followed by plasmid preparation and purification, and then was confirmed by the analysis of dual- endonuclease digestion and sequencing.4. Hepatocellular carcinoma cell SMMC7721 and macrophage U937 cell culture: add a high-glucose DMEM medium with 10% FBS, medium should be changed every two days, cells should be passaged when there were filled with 90% of the cultrue plates.5. The confirmed recombinant pEGFP-N1-Tim-3 was transfected into hepatocellular carcinoma cell SMMC7721 and macrophage U937 cell by liposome method to observe the expression of GFP under immunofluorescence microscope.Results:1. The Tim-3 cDNA was particularly amplified with primers from the total RNA of the PBMC of the health person. Sequencing demonstrate that pGEM-T-Tim-3 recombinant plasmid was successfully constructed.2. The recombinant pEGFP-N1-Tim-3 plasmid was confirmed by analysis of restriction endonuclease digestion and sequencing. The inserted Tim-3 cDNA segment in the vector was sense orientation and was consistent with that of the published data (GenBank access NM₀32782.3).3. The fusion gene could be effectively transcripted and translated. The expression of GFP in pEGFP-N1-Tim-3 transfected hepatocellular carcinoma cell SMMC7721 and macrophage U937 cell was observed and located at the membrane of the cell.Conclusion:1. The Tim-3 cDNA was particularly amplified with primers from the total RNA of the PBMC of the health person. Sequencing demonstrate that pGEM-T-Tim-3 recombinant plasmid was successfully constructed.2. The pGEM-T-Tim-3 recombinant plasmid was purified and digested with Bgl â…¡and Salâ… ,then the Tim-3 was inserted into the Bglâ…¡and Salâ… sites of the eukaryotic expression plasmid pEGFP-N1.Sequencing demonstrate that pEGFP-N1- Tim-3 recombinant plasmid was successfully constructed.3. The fusion gene could effectively transcripted and translated. The expression of GFP in pEGFP-N1-Tim-3 transfected SMMC7721 cell and macrophage U937 was observed and located at the membrane of the cell. It was consistent with the Tim-3 was the type I membrane protein and demonstrate that the fusion gene could be effectively translated.4. We have successfully constructed pEGFP-N1-Tim-3 eukaryotic expression plasmid, it could be expressed in several eukaryotic cells. The newly constructed recombinant vector is useful for follow-up identification of the function of Tim-3 protein in different cells, and it would be a new target for treat diseases.