Hamed Salehi-Lalemarzi1, Dariush Shanehbandi2, Farzaneh Shafaghat3, Hajar Abbasi-Kenarsari4, Behzad Baradaran5, Ali Akbar Movassaghpour6, Tohid Kazemi7. 1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, International Branch of Aras, Tabriz University of Medical Sciences, Tabriz, Iran. ; Students' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Department of Immunology, International Branch of Aras, Tabriz University of Medical Sciences, Tabriz, Iran. 4. Drug Applied Research Center and Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 5. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Drug Applied Research Center and Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 6. Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 7. Drug Applied Research Center and Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. ; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, International Branch of Aras, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
PURPOSE: PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. METHODS: CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. RESULTS: 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. CONCLUSION: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins.
PURPOSE:PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for humanCD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. METHODS:CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. RESULTS: 2235 bp specific band was aligned completely to humanCD31 reference sequence in NCBI database. Transient and stable expression of humanCD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. CONCLUSION: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against humanCD31, with no need for purification of recombinant proteins.
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