Producing Recombinant HEK293 T-cells with High Expression of T-cell Immunoglobulin and Mucin Domain-3 (Tim3) Protein

Document Type : Original Article (s)

Authors

1 MSc Student, Department of Genetics and Molecular Biology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

2 Assistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 PhD Student, Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

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. 

Keywords

References

  1. Meyers JH, Sabatos CA, Chakravarti S, Kuchroo VK. The TIM gene family regulates autoimmune and allergic diseases. Trends Mol Med 2005; 11(8): 362-9.
  2. Anderson AC, Anderson DE. TIM-3 in autoimmunity. Curr Opin Immunol 2006; 18(6): 665-9.
  3. Kuchroo VK, Dardalhon V, Xiao S, Anderson AC. New roles for TIM family members in immune regulation. Nat Rev Immunol 2008; 8(8): 577-80.
  4. Li S, Peng D, He Y, Zhang H, Sun H, Shan S, et al. Expression of TIM-3 on CD4+ and CD8+ T cells in the peripheral blood and synovial fluid of rheumatoid arthritis. APMIS 2014; 122(10): 899-904.
  5. Yeung MY, McGrath M, Najafian N. The emerging role of the TIM molecules in transplantation. Am J Transplant 2011; 11(10): 2012-9.
  6. Nagahara K, Arikawa T, Oomizu S, Kontani K, Nobumoto A, Tateno H, et al. Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions. J Immunol 2008; 181(11): 7660-9.
  7. Yan J, Zhang Y, Zhang JP, Liang J, Li L, Zheng L. Tim-3 expression defines regulatory T cells in human tumors. PLoS One 2013; 8(3): e58006.
  8. Rodriguez-Manzanet R, DeKruyff R, Kuchroo VK, Umetsu DT. The costimulatory role of TIM molecules. Immunol Rev 2009; 229(1): 259-70.
  9. Lee J, Phong B, Egloff AM, Kane LP. TIM polymorphisms--genetics and function. Genes Immun 2011; 12(8): 595-604.
  10. Su EW, Lin JY, Kane LP. TIM-1 and TIM-3 proteins in immune regulation. Cytokine 2008; 44(1): 9-13.
  11. Vega-Carrascal I, Reeves EP, McElvaney NG. The role of TIM-containing molecules in airway disease and their potential as therapeutic targets. J Inflamm Res 2012; 5: 77-87.
  12. Chae SC, Park YR, Shim SC, Yoon KS, Chung HT. The polymorphisms of Th1 cell surface gene Tim-3 are associated in a Korean population with rheumatoid arthritis. Immunol Lett 2004; 95(1): 91-5.
  13. Kuchroo VK, Umetsu DT, DeKruyff RH, Freeman GJ. The TIM gene family: emerging roles in immunity and disease. Nat Rev Immunol 2003; 3(6): 454-62.
  14. Pan HF, Zhang N, Li WX, Tao JH, Ye DQ. TIM-3 as a new therapeutic target in systemic lupus erythematosus. Mol Biol Rep 2010; 37(1): 395-8.
  15. Li X, Zhao YQ, Li CW, Yuan FL. T cell immunoglobulin-3 as a new therapeutic target for rheumatoid arthritis. Expert Opin Ther Targets 2012; 16(12): 1145-9.
  16. Freeman GJ, Casasnovas JM, Umetsu DT, DeKruyff RH. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunol Rev 2010; 235(1): 172-89.
  17. McMahan RH, Golden-Mason L, Nishimura MI, McMahon BJ, Kemper M, Allen TM, et al. Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J Clin Invest 2010; 120(12): 4546-57.
  18. Sakuishi K, Jayaraman P, Behar SM, Anderson AC, Kuchroo VK. Emerging Tim-3 functions in antimicrobial and tumor immunity. Trends Immunol 2011; 32(8): 345-9.
  19. Ngiow SF, von SB, Akiba H, Yagita H, Teng MW, Smyth MJ. Anti-TIM3 antibody promotes T cell IFN-gamma-mediated antitumor immunity and suppresses established tumors. Cancer Res 2011; 71(10): 3540-51.
  20. Block MS, Markovic SN. The tumor/immune interface: clinical evidence of cancer immunosurveillance, immunoediting and immunosubversion. Am J Immunol 20091: 5(1): 29-49.
  21. Zhuang X, Zhang X, Xia X, Zhang C, Liang X, Gao L, et al. Ectopic expression of TIM-3 in lung cancers: a potential independent prognostic factor for patients with NSCLC. Am J Clin Pathol 2012; 137(6): 978-85.
  22. Chae SC, Park YR, Lee YC, Lee JH, Chung HT. The association of TIM-3 gene polymorphism with atopic disease in Korean population. Hum Immunol 2004; 65(12): 1427-31.
  23. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002; 415(6871): 536-41.
  24. McIntire JJ, Umetsu SE, Akbari O, Potter M, Kuchroo VK, Barsh GS, et al. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol 2001; 2(12): 1109-16.
  25. Chen Z, Qing J, Qin G, Hu L. Construction and characterization of bifunctional TIM-3-EGFP fusion proteins. Protein Expr Purif 2012; 86(1): 1-6.
Journal of Isfahan Medical School
Volume 32, Issue 317 - Serial Number 317
November and December 2015
Pages 2312-2323

Files

History

  • Receive Date: 19 January 2015
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

Share

How to cite

Statistics