مجله دانشکده پزشکی اصفهان (Mar 2015)
Producing Recombinant HEK293 T-cells with High Expression of T-cell Immunoglobulin and Mucin Domain-3 (Tim3) Protein
Abstract
Background: T-cell immunoglobulin and mucin domain-3 (Tim3) is known as a marker of cell surface of T-helper-1 (Th1) cell and has a key role in many diseases with cell-mediated immunity. Over expression of Tim3 has reported in autoimmune and atopic diseases. Though, many studies done about this inhibitory help protein molecules, but yet need more research. In research project on Tim3, native protein with proper post translation modification should be provided. In this study, Tim3 was expressed on the surface of HEK293 T-cell line. Methods: Tim3 expression cassette was amplified from cDNA clone EX-W2682-M67 via polymerase chain reaction (PCR). The PCR product and pHygro plasmid were digested by NheI and MluI restriction enzymes. The linearized pHygro and digested PCR product were ligated together with T4 DNA ligase and transformed into Escherichia coli TOP 10 F'. The resulted pH-Tim3 plasmid was linearized with NheI and transfected into 293T cell line. The transfected cells were positive selected with hygromycin and their genomic DNA was extracted and PCR was done on them to amplify complementary DNA (cDNA) of Tim3. Also, protein expression levels were assessed via flow cytometry. Findings: The result of PCR on selected cells confirmed integratioin of cDNA clone of Tim3 in genomic DNA. Based on flow cytometry, about 88% of cells were expressed Tim3 sharply. Conclusion: In order to understand the role and mechanism of Tim3 protein, we need a large amount of purified native Tim3. Prokaryotic expression systems are simpler and cheaper than eukaryotic expression system, but they are not proper system for expression of proteins that modified after translation. Expression of membrane proteins like Tim3 on the surface of cell has even more native conformation in comparison with recombinant Tim3. The cells display Tim3 could be used in antibody, nanobody or aptamer production projects. In this project, we constructed a 293T cell which overexpressed Tim3 on its surface compared to untransfected ones.
