Development of a stable cell line, overexpressing human T-cell immunoglobulin mucin 1


Ebrahimi M., Kazemi T., Ganjalikhani-Hakemi M., Majidi J., Khanahmad H., Rahimmanesh I., ...More

Iranian Journal of Biotechnology, vol.13, no.4, 2015 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 13 Issue: 4
  • Publication Date: 2015
  • Doi Number: 10.15171/ijb.1350
  • Journal Name: Iranian Journal of Biotechnology
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Keywords: Cloning, Gene expression, HEK 293T, Immunogenic source, TIM-1
  • Istanbul Medipol University Affiliated: No

Abstract

Background: Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore,targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives: In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods: The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results: The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions: Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1.