Stable Expression of Recombinant RhD Antigen Isolated from Cord Blood in K562 Cell Line
Journal Title: Iranian Journal of Blood and Cancer - Year 2009, Vol 1, Issue 2
Abstract
Background: The Rh antigens are expressed as parts of a protein complex in the RBC membrane. This complex is a tetramer, consisting of two molecules of RhAG and two molecules of Rh proteins. To express RhD in RBC membrane, expression of RhAG is essential. This co-expression only occurs in the erythroid lineage. K562 cell line has an erythroid lineage. Materials and Methods: Cord blood was used as a source of RHD gene in which nucleated RBCs are rich. Mononuclear cells were isolated using Ficol method. RNA was extracted by trizol followed by cDNA synthesis. RHD gene was isolated with specific primers. The RHD cDNA was ligated to pcDNA3.1 vector and cloned into E. coli. The recombinant pcDNA-RHD construct was transfected to K562 cell line. Stable cells expressing RhD were selected in the presence of geneticin. RT-PCR and western blot analysis were performed to detect recombinant RhD. Results: Stable cells expressing recombinant RhD were established. RT-PCR results showed exogenous expression of recombinant RhD which was further confirmed by western blot analysis. Conclusion: Overall, our results revealed that K562 is suitable for expression of RhD. The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein.
Authors and Affiliations
Mehryar Habibi Roudkenar, Mahshid Mohammadipour, Arezou Oodi, Raheleh Halabian, Naser Amirizadeh, Nasser Masrouri, Amaneh Mohammadi Roshandeh, Kazem parivar, Houri Rezvan
Keywords
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