构建稳定表达病毒相关RNA的真核细胞株及其对抗肿瘤免疫促凋亡分子的影响
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摘要
目的:构建稳定表达病毒相关RNA(virus-associated RNA,VA RNA)的293F-VA细胞株,并探讨其对靶向HER2阳性肿瘤的免疫促凋亡分子表达水平的影响。方法:采用PCR方法,扩增含VA RNA的功能序列,克隆入Piggy Bac转座系统质粒,将VA RNA编码序列转入293F细胞中,经过抗性筛选和流式细胞仪鉴定,得到293F-VA细胞株。将萤光素酶、红色荧光蛋白及免疫促凋亡分子编码质粒分别转入293F-VA细胞株或亲本293F细胞株,通过萤光素酶活力检测和Western Blot实验,比较两种细胞株对外源目的蛋白表达水平的影响。结果:成功构建了Piggy Bac-VA质粒。共转染Piggy Bac-VA质粒可以明显提高细胞内Fluc蛋白的表达水平(P <0. 05)。VA RNA编码序列通过Piggy Bac转座系统转入293F细胞中,获得293F-VA细胞株。与293F亲本细胞相比,293F-VA细胞株将萤火虫萤光素酶Fluc蛋白的表达水平提高363. 9%(P<0. 05);对海肾萤光素酶Rluc蛋白及红色荧光蛋白m Cherry的表达均表现出3倍左右的提高(P <0. 05)。293F-VA细胞株不仅能够提高免疫促凋亡分子e23-Fc-Fdt-t Bid在细胞内的表达水平,并且显著增加目的蛋白向细胞外分泌的水平,较293F亲本细胞提高4. 2倍(P <0. 05)。结论:建立了稳定表达VA RNA的细胞株293F-VA,该细胞株能够显著提高抗肿瘤免疫促凋亡分子的表达水平。
Objective: To discuss the influence on anti-tumor immunoproapoptotin in 293 F 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 Piggy Bac transposon system. The VA RNA coding sequence was transfected into 293 F cells using the Piggy Bac 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 293 F 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 Piggy Bac transposon vector,and constructed into Piggy Bac-VA plasmid. Piggy Bac-VA plasmid and pc DNA3. 1-Fluc plasmid were transiently transfected into 293 F 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 293 F cells by the Piggy Bac transposon system. After puromycin resistance screening and validation by fluorescence microscopy and flow cytometry,almost100% of the 293F-VA cells were EGFP-positive. The intracellular protein expression capacity of pc DNA3. 1-Fluc plasmid was increased by 363. 9% in the 293F-VA cell line( P < 0. 05). The intracellular protein expression capacity of pc DNA3. 1-Rluc plasmid was increased by 302. 2%( P < 0. 05). The intracellular protein expression capacity of plasmid pc DNA3. 1-m Cherry was increased by 3 fold( P < 0. 05). The intracellular expression level of the immunoproapoptotin plasmid pc DNA3. 1-e23-Fc-Fdt-t Bid 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.
Objective: To discuss the influence on anti-tumor immunoproapoptotin in 293 F 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 Piggy Bac transposon system. The VA RNA coding sequence was transfected into 293 F cells using the Piggy Bac 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 293 F 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 Piggy Bac transposon vector,and constructed into Piggy Bac-VA plasmid. Piggy Bac-VA plasmid and pc DNA3. 1-Fluc plasmid were transiently transfected into 293 F 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 293 F cells by the Piggy Bac transposon system. After puromycin resistance screening and validation by fluorescence microscopy and flow cytometry,almost100% of the 293F-VA cells were EGFP-positive. The intracellular protein expression capacity of pc DNA3. 1-Fluc plasmid was increased by 363. 9% in the 293F-VA cell line( P < 0. 05). The intracellular protein expression capacity of pc DNA3. 1-Rluc plasmid was increased by 302. 2%( P < 0. 05). The intracellular protein expression capacity of plasmid pc DNA3. 1-m Cherry was increased by 3 fold( P < 0. 05). The intracellular expression level of the immunoproapoptotin plasmid pc DNA3. 1-e23-Fc-Fdt-t Bid 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.
引文
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[3]Yan B,Ouyang Q,Zhao Z,et al.Potent killing of HBV related hepatocellular carcinoma by a chimeric protein of anti-HBsAg singlechain antibody and truncated Bid[J].Biomaterials,2013,34(20):4880-4889.
[4]Demonte D,Dundas CM,Park S.Expression and purification of soluble monomeric streptavidin in escherichia coli[J].Appl Microbiol Biotechnol,2014,98(14):6285-6295.
[5]Farrell PJ,Balkow K,Hunt T,et al.Phosphorylation of initiation factor el F-2 and the control of reticulocyte protein synthesis[J].Cell,1977,11(1):187-200.
[6]Vachon VK,Conn GL.Adenovirus VA RNA:An essential pro-viral non-coding RNA[J].Virus Res,2016,212(2):39-52.
[7]O'Malley RP,Mariano TM,Siekierka J,et al.A mechanism for the control of protein synthesis by adenovirus VA RNAI[J].Cell,1986,44(3):391-400.
[8]Deckmann K,Krasteva-Christ G,Rafiq A,et al.Cholinergic urethral brush cells are widespread throughout placental mammals I[J].Int Immunopharmacol,2015,29(1):51-56.
[9]Dzananovic E,Patel TR,Chojnowski G,et al.Solution conformation of adenovirus virus associated RNA-I and its interaction with PKR[J].J Struct Biol,2014,185(1):48-57.
[10]Alhammad RM,Dronca RS,Kottschade LA.Brachial plexus neuritis associated with anti-programmed cell death-1 antibodies:Report of 2 cases[J].Mayo Clin Proc Innov Qual Outcomes,2017,1(2):192-197.
[11]Pastor M,Johnen S,Harmening N,et al.The antibiotic-free pFAR4 vector paired with the sleeping beauty transposon system mediates efficient transgene delivery in human cells[J].Mol T-her Nucleic Acids,2018,1(11):57-67.
[12]Wu Y,Zhang TY,Fang LL,et al.Sleeping Beauty transposonbased system for rapid generation of HBV-replicating stable cell lines[J].J Virol Methods,2016,234:96-100.
[13]Zhao S,Jiang E,Chen S,et al.Piggy Bac transposon vectors:The tools of the human gene encoding[J].Transl Lung Cancer Res,2016,5(1):120-125.
[14]Woodard LE,Wilson MH.Piggy Bac-ing models and new therapeutic strategies[J].Trends Biotechnol,2015,33(9):525-533.
[15]Savage P.Approvals in 2016:Cost-benefit challenges of new anticancer agents[J].Nat Rev Clin Oncol,2017,14(3):133-134.
[16]Bire S,Ishac N,Rouleux-Bonnin F.In vitro synthesis,delivery,and bioavailability of exogenous mrna in gene transfer mediated by piggybac transposition[J].Methods Mol Biol,2016,14(28):187-217.
[17]Zoltán Ivics.Endogenous transposase source in human cells mobilizes piggybac transposons[J].Mol Ther,2016,24(5):851-854.
[18]Chadda R,Robertson JL.Measuring membrane protein dimerization equilibrium in lipid bilayers by single-molecule fluorescence microscopy[J].Methods Enzymol,2016,581:53-82.
[19]Shuen WH,Kan R,Yu Z,et al.Novel lentiviral-inducible transgene expression systems and versatile single-plasmid reporters for in vitro and in vivo cancer biology studies[J].Cancer Gene Ther,2015,22(4):207-214.
[20]Ramirez-Mata A,Pacheco MR,Moreno SJ,et al.Versatile use of azospirillum brasilense strains tagged with egfp and m Cherry genes for the visualization of biofilms associated with wheat roots[J].Microbiol Res,2018,215:155-163.
[21]Mills CE,Michaud Z,Olsen BD,et al.Elastin-like polypeptide(ELP)charge influences self-assembly of ELP-m Cherry fusion proteins[J].Biomacromolecules,2018,19(7):2517-2525.
[22]Ouyang Q,Yan B,Li A,et al.Construction of humanized antiHER2 single-chain variable fragments(husFvs)and achievement of potent tumor suppression with the reconstituted husFvFdt-t Bid immunoapoptotin[J].Biomaterials,2018,17(8):170-182.