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Title: Cell surface display of rabbit MCP1 on human embryonic kidney 293T cell line
Authors: Rahimmanesh, Ilnaz
Khanahmad, Hossein
Boshtam, Maryam
Kouhpayeh, Shirin
Hejazi, Zahra
Keywords: Cell surface display;Rabbit MCP-1;Expression vector;Gene expression;Transfection;Transformation
Issue Date: Jul-2017
Publisher: NISCAIR-CSIR, India
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.
Page(s): 284-288
ISSN: 0975-0967 (Online); 0972-5849 (Print)
Appears in Collections:IJBT Vol.16(3) [July 2017]

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