Author/Authors :
Salehi-Lalemarzi، Hamed نويسنده Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. , , Shanehbandi، Dariush نويسنده Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. , , Shafaghat، Farzaneh نويسنده Department of Immunology, International Branch of Aras, Tabriz University of Medical Sciences , , Abbasi-Kenarsari، Hajar نويسنده Immunology Research Center, Tabriz University of Medical Sciences, Department of Immunology, School of Medicine, Tabriz University of Medical SciencesStudents Research Committee, Tabriz University of Medical Sciences , , Baradaran، Behzad نويسنده Department of Immunology, Tabriz University of Medical Sciences, Immunology research center, Tabriz, Iran Baradaran, Behzad , Movassaghpour، Ali Akbar نويسنده Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. , , Kazemi، Tohid نويسنده Immunology Research Center, 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 ?g of recombinant construct and 6 ?l 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.
