|Table of Contents|

Construction of stably expressing virus-associated RNA cell and the influence on anti-tumor immunoproapoptotin expression

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

Issue:
2019 03
Page:
367-371
Research Field:
Publishing date:

Info

Title:
Construction of stably expressing virus-associated RNA cell and the influence on anti-tumor immunoproapoptotin expression
Author(s):
Li Ang1Lun Xinxin2Chen Yuankun2Yang Tian1Yang Angang1Yan Bo3
1.Department of Immunology,Air Force Medical University,Shaanxi Xi'an 710032,China;2.School of Labratory Medicine,Xinxiang Medical University,Henan Xinxiang 453000,China;3.Department of Biochemistry and Molecular Biology,Air Force Medical University,Shaanxi Xi'an 710032,China.
Keywords:
virus-associated RNAmammalian cell protein expression system293F celltumor targeting therapy
PACS:
R730.54
DOI:
10.3969/j.issn.1672-4992.2019.03.002
Abstract:
Objective:To discuss the influence on anti-tumor immunoproapoptotin in 293F cell line by constructing a 293F-VA cell line stably expressing virus-associated RNA(VA RNA).Methods:The functional sequence encoding VA RNA was amplified by PCR and cloned into the plasmid of PiggyBac transposon system.The VA RNA coding sequence was transfected into 293F cells using the PiggyBac transposon system,and was identified by resistance screening and flow cytometry to obtain 293F-VA cell line.The plasmids encoding luciferase,red fluorescent protein and immunoproapoptotin were transfected into 293F-VA cell line or parental 293F cell line respectively,and the two cell lines were compared for expression level of exogenous protein by luciferase activity assay and Western Blot.Results:The coding sequence of VA RNA was amplified by PCR,cloned into PiggyBac transposon vector,and constructed into PiggyBac-VA plasmid.PiggyBac-VA plasmid and pcDNA3.1-Fluc plasmid were transiently transfected into 293F cells.The intracellular protein expression of Fluc was significantly increased compared with the control group(P<0.05).The VA RNA coding sequence was transfected into 293F cells by the PiggyBac transposon system.After puromycin resistance screening and validation by fluorescence microscopy and flow cytometry,almost 100% of the 293F-VA cells were EGFP-positive.The intracellular protein expression capacity of pcDNA3.1-Fluc plasmid was increased by 363.9% in the 293F-VA cell line(P<0.05).The intracellular protein expression capacity of pcDNA3.1-Rluc plasmid was increased by 302.2%(P<0.05).The intracellular protein expression capacity of plasmid pcDNA3.1-mCherry was increased by 3 fold (P<0.05).The intracellular expression level of the immunoproapoptotin plasmid pcDNA3.1-e23-Fc-Fdt-tBid was increased by 1.2-fold (P<0.05),and the secretion level was increased by 4.2-fold (P<0.05).Conclusion:A stable cell line 293F-VA expressing VA RNA was established.The cell line could significantly improve the expression of anti-tumor immunoproapoptotin.

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Memo

Memo:
National Natural Science Foundation of China(No.81630069);国家自然科学基金重点项目(编号:81630069)
Last Update: 2018-12-29