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[TM]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