Construction and Identification of HEK-293 Cell Lines with Stable Overexpression of CXCR2 Receptor

ZHANG Jie¹, REN Guangming², MA Wenbing², GE Zhiqiang¹, ZHAN Yiqun², YIN Ronghua²*, YANG Xiaoming^1,2*

(¹Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China; ²Beijing Institute of Lifeomics, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 102206, China)

Abstract:

CXCR2 (the CXC chemokine receptor 2)-mediated neutrophil chemotaxis plays an important role in the process of bacterial infection, chronic inflammation and tumor development, but the regulation of the activation of CXCR2 receptor and downstream signals are not fully understood at present. In this study, Flag-CXCR2 was firstly amplified from Flag-CXCR2-Tango plasmid, then inserted it into pCDH-MCS-T2A-Neo-MSCV expression vector. The construction was confirmed by sequencing and the expression of Flag-CXCR2 was detected by Western blot. Then, the lentivirus particles expressing Flag-CXCR2 were generated in HEK-293T cells and transduced into HEK-293 cells. After screening with neomycin, Western blot and immunofluorescence detection showed that Flag-CXCR2 stably expressed on HEK-293 cell membrane. Finally, the overexpression of Flag-CXCR2 cell line was stimulated with IL-8 (interleukin-8). The activation of signal pathways downstream of CXCR2 and F-actin assembly were detected.

CSTR: 32200.14.cjcb.2020.08.0003