Expression and purification of mIL-21-hIgGFc fusion protein in 293E cells and its effects on CD8+T cell phenotype
10.3969/j.issn.1000-484X.2014.07.005
- VernacularTitle:mIL-21-hIgGFc融合蛋白在293 E细胞中的表达纯化及其对CD8＋T细胞表型的影响
- Author:
Qibin HUANG
;
Mingyue LIU
;
Shaoyue FU
;
Qiao XING
;
Xiaoqi LIU
;
Shengdian WANG
;
Faping YI
- Publication Type:Journal Article
- Keywords:
Interleukin-21(IL-21);
293E cells;
Expression and purification;
CD8+T cells
- From:
Chinese Journal of Immunology
2014;(7):884-887,892
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To express recombinant protein mIL-21-hIgGFc in 293E cells,and investigate its effect on CD8+T cell.Methods:Total RNA was extracted from the mouse spleen cells ,and then IL-21 gene was amplified by RT-PCR and inserted into expression vector PTT3-hIgGFc.PTT3-mIL-21-hIgGFc were transfected into 293E cells by calcium phosphate method.The supernatants were collected at 48 hours and 72 hours and concentrated by MOLLIPORE Labscale TM TFF system ( 5 kD membrane ).The mIL-21-hIgGFc fusion protein was purified with HiTrap TM Protein G column.The protein was quantified by SDS-PAGE and ELISA.The biological activity of the protein was determined by detecting the change of the phenotypes of CD 8+ T cells treated with the protein.Results: The constructed recombinant plasmid PTT 3-mIL-21-Fc was confirmed by sequencing.PTT3-mIL-21-Fc was transfected into 293E cells,mIL-21-Fc protein in culture supernatant was collected after 48 hours and 72 hours.The protein in cell su-pernatant reached a concentration of 787 ng/ml which was determined by ELISA.The protein was purified by Protein G chromatography column.P1A-specific T cells were treated with mIL-21-hIgGFc, and found that the CD44low CD62Lhi CD8+ population increased compared to the control.Conclusion:We built PTT3-mIL-21-hFc recombinant plasmid, expressed mIL21-hFc fusion protein in 293E cells,and purified by Protein G column.By treating mIL-21-hFc ,the antigen-primed CD8+T cells prefer to differentiate into CD44low CD62Lhi CD8+T cells which had been reported as a memory stem phenotype .This protein may be used to improve the effectness of adoptive T cell cancer therapy.
