Cell Surface Display of Rabbit Mcp1 on Human Embryonic Kidney 293T Cell Line



Rahimmanesh I¹ ; Khanahmad H¹ ; Boshtam M² ; Kouhpayeh S³ ; Hejazi Z¹ Show Affiliations
Source: Indian Journal of Biotechnology Published:2017

Abstract

As atherosclerosis is a prevalent non-communicable disease, and yet, no definitive medical treatment found for it, Trapping MCP1 as a key factor in inflammation could be effective. Therefore, we decided to display rabbit MCP-1 (R-MCP1) on human embryonic kidney 293T cell line surface. Firstly, R-MCP1 plasmid (pR-MCP1) containing kappa chain signal sequence, R-MCP1 sequence and PDGFR intra membrane domain was constructed. The delivered pR-MCP1 was transformed in E. coli TOP10F', and the resulted clones were assessed by PCR and digestion. After linearizing pR-MCP1 by BglII, HEK cells were transfected by them. MCP1 gene integration and expression was confirmed at RNA and protein levels by real- time PCR and flow cytometry, respectively. PCR product gel electrophoresis on genomic DNA of transfected HEK cells showed a 737 bp band. Based on real- time PCR results, We observed R-MCP1 gene expression significantly increased in transfected cells (272.26 ± 37.32) compare to untransfected HEK 293T cells (2.67 ± 0.12) (p = 0.001). The results of flow cytometry showed that about 85% of transfected cells were positive and express R-MCP1. Therefore, cell surface display of R-MCP1 has successfully been performed and the produced cells can be used in future research to prepare diagnostic and therapeutic agents like aptamers.