Mina Ebrahimi1,2,3, Tohid Kazemi2, Mazdak Ganjalikhani-Hakemi4, Jafar Majidi2, Hossein Khanahmad5, Ilnaz Rahimmanesh5, Vida Homayouni4, Shirin Kohpayeh4. 1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Tabriz University of Medical Sciences, Tabriz, Iran. 4. Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. 5. Department of Genetic and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
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.
BACKGROUND: Recent researches have demonstrated that humanT-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 HumanEmbryonic 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 humanTIM-1.
Authors: Andrew S Kondratowicz; Nicholas J Lennemann; Patrick L Sinn; Robert A Davey; Catherine L Hunt; Sven Moller-Tank; David K Meyerholz; Paul Rennert; Robert F Mullins; Melinda Brindley; Lindsay M Sandersfeld; Kathrina Quinn; Melodie Weller; Paul B McCray; John Chiorini; Wendy Maury Journal: Proc Natl Acad Sci U S A Date: 2011-05-02 Impact factor: 11.205
Authors: Hyun-Hee Lee; Everett H Meyer; Sho Goya; Muriel Pichavant; Hye Young Kim; Xia Bu; Sarah E Umetsu; Jennifer C Jones; Paul B Savage; Yoichiro Iwakura; Jose M Casasnovas; Gerardo Kaplan; Gordon J Freeman; Rosemarie H DeKruyff; Dale T Umetsu Journal: J Immunol Date: 2010-10-01 Impact factor: 5.422