|Table of Contents|

Construction of human PD-L1 eukaryotic expression plasmid and screening of single overexpression stable colon cancer cell line

Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:
2018 06
Page:
843-847
Research Field:
Publishing date:

Info

Title:
Construction of human PD-L1 eukaryotic expression plasmid and screening of single overexpression stable colon cancer cell line
Author(s):
Mao Chengyi1; Du Juan1; Ma Yu1; Li Xiangyun2; Ao Luoquan2; Xu Xiang2; Xiao Hualiang1
1.Department of Pathology;2.State Key Laboratory of Trauma,Burns and Combined Injury,Department 1,Institute of Surgery Research,Daping Hospital,Third Military Medical University,Chongqing 400042,China.
Keywords:
PD-L1; recombinant plasmid vector; single overexpression stable colon cancer cell line
PACS:
R735.35
DOI:
10.3969/j.issn.1672-4992.2018.06.007
Abstract:
Objective:To construct the human PD-L1 recombinant plasmid vector and screen the single stable colon cancer cell line over-expressing human PD-L1.Methods:Human PD-L1 gene was cloned by RT-PCR from total RNA in HCT116 cells.Gene fragments were linked to HA labeled pcDNA3 with Neo selecting marker to transfect HCT116 cells.Cells were then dispersed to 96-well plates and screened by G418 for 2 weeks.PD-L1 expression level in the monoclone cell lines were identified by Western Blot.The effects of PD-L1 expression level on cell growth migration and colony formation ability were evaluated by MTS，Transwell and soft agar assay.We further elucidated the partial mechanism leading to those phenomena by overexpression of cap-dependent luciferase reporter gene assay.Results:The human PD-L1 eukaryotic expression vector and monoclone PD-L1 over-expressing stable cell lines were successfully constructed.Preliminary function study showed that PD-L1 could promote colon cancer cell growth,migration and colony formation capacity.PD-L1 upregulated cap-dependent luciferase expression by 3~4 folds which indicated that the mechanism was partially from elevated cap-dependent protein expression in the PD-L1 high-expression cell lines.Conclusion:We generated the human PD-L1 recombinant plasmid vector and single HCT116 stable cell line over-expressing human PD-L1 which can be used for further PD-L1 functional study.

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Last Update: 2018-01-29