ERO1α慢病毒过表达载体的构建及其在RAW264.7细胞中稳定表达
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摘要
[目的]构建小鼠ERO1α基因慢病毒过表达载体,并筛选其在RAW264.7细胞中稳定表达。[方法]利用PCR方法扩增ERO1α基因的CDS区并测序;将目的片段亚克隆到慢病毒过表达载体pCD513B-1上,构建pCD513B-ERO1α慢病毒过表达载体;通过病毒包装产生慢病毒并转导RAW264.7细胞;转导后的细胞进行嘌呤霉素抗性筛选,获得稳定表达的细胞;利用RT-qPCR和Western blot方法检测ERO1α的表达效果。[结果]ERO1α慢病毒过表达载体构建成功,病毒滴度为5×10~6～10×10~6 TU/mL;慢病毒成功转导RAW264.7细胞并且稳定高表达。[结论]成功构建ERO1α慢病毒过表达载体,并在RAW264.7细胞中稳定高表达,为进一步研究ERO1α在免疫系统中的功能提供了技术支持。
[Objective]To construct ERO1α recombinant lentiviral overexpression vector,and to stably express in RAW264.7 macrophages.[Method]The CDS sequences of mouse ERO1α were amplified by PCR.The fragment of ERO1α was cloned into the lentiviral shuttle vector pCD513 B-1.The recombinant lentivirus was packaged and transduced into RAW264.7 macrophages.ERO1α mRNA and protein expression were examined using real-time quantitative PCR(RT-qPCR) and western blot.[Result]The ERO1α recombinant lentiviral overexpression vector was successfully constructed,and the final titer was 5×10~6-10×10~6 TU/mL.After transducing into RAW264.7 macrophages and puromycin screening,RT-qPCR and Western blot results showed that the overexpression vector significantly increased the expression of exogenous ERO1α.[Conclusion]The recombinant lentiviral vector is successfully constructed and stably expressed in RAW264.7 macrophages,which is a promising tool for studying the function of ERO1α in the immune system.
[Objective]To construct ERO1α recombinant lentiviral overexpression vector,and to stably express in RAW264.7 macrophages.[Method]The CDS sequences of mouse ERO1α were amplified by PCR.The fragment of ERO1α was cloned into the lentiviral shuttle vector pCD513 B-1.The recombinant lentivirus was packaged and transduced into RAW264.7 macrophages.ERO1α mRNA and protein expression were examined using real-time quantitative PCR(RT-qPCR) and western blot.[Result]The ERO1α recombinant lentiviral overexpression vector was successfully constructed,and the final titer was 5×10~6-10×10~6 TU/mL.After transducing into RAW264.7 macrophages and puromycin screening,RT-qPCR and Western blot results showed that the overexpression vector significantly increased the expression of exogenous ERO1α.[Conclusion]The recombinant lentiviral vector is successfully constructed and stably expressed in RAW264.7 macrophages,which is a promising tool for studying the function of ERO1α in the immune system.
引文
[1] POLLARD M G,TRAVERS K J,WEISSMAN J S.ERO1p:A novel and ubiquitous protein with an essential role in oxidative protein folding in the endoplasmic reticulum[J].Molecular cell,1998,1(2):171-182.
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[3] ARAKI K,NAGATA K.Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway[J].The journal of biological chemistry,2011,286(37):32705-32712.
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[5] MAY D,ITIN A,GAL O,et al.ERO1-L α plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia:Implication for cancer[J].Oncogene,2005,24:1011-1020.
[6] KUKITA K,TAMURA Y,TANAKA T,et al.Cancer-associated oxidase ERO1-α regulates the expression of MHC class I molecule via oxidative folding[J].Journal of immunology,2015,194(10):4988-4996.
[7] TANAKA T,KAJIWARA T,TORIGOE T,et al.Cancer-associated oxidoreductase ERO1-α drives the production of tumor-promoting myeloid-derived suppressor cells via oxidative protein folding[J].Journal of immunology,2015,194(4):2004-2010.
[8] 陈风雷.小鼠Zhangfei基因过表达和shRNA干扰慢病毒载体的构建和鉴定[D].杨凌:西北农林科技大学,2013:42-43.
[9] CHEN F L,LIN P F,LI X,et al.Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells[J].Plasmid,2014,76:24-31.
[10] CHEN F L,LI Q,ZHANG J Y,et al.Silencing effect of lentiviral vectors encoding shRNA of Herp on endoplasmic reticulum stress and inflammatory responses in RAW 264.7 macrophages[J].Genetics and molecular research,2015,14(4):17587-17598.
[11] RASCHKE W C,BAIRD S,RALPH P,et al.Functional macrophage cell lines transformed by Abelson leukemia virus[J].Cell,1978,5(1):261-267.
[12] BLESCH A.Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer[J].Methods,2004,33(2):164-172.
[2] FRAND A R,KAISER C A.ERO1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum[J].Molecular cell,1999,4(4):469-477.
[3] ARAKI K,NAGATA K.Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway[J].The journal of biological chemistry,2011,286(37):32705-32712.
[4] KUTOMI G,TAMURA Y,TANAKA T,et al.Human endoplasmic reticulum oxidoreductin 1-α is a novel predictor for poor prognosis of breast cancer[J].Cancer Sci,2013,104(8):1091-1096.
[5] MAY D,ITIN A,GAL O,et al.ERO1-L α plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia:Implication for cancer[J].Oncogene,2005,24:1011-1020.
[6] KUKITA K,TAMURA Y,TANAKA T,et al.Cancer-associated oxidase ERO1-α regulates the expression of MHC class I molecule via oxidative folding[J].Journal of immunology,2015,194(10):4988-4996.
[7] TANAKA T,KAJIWARA T,TORIGOE T,et al.Cancer-associated oxidoreductase ERO1-α drives the production of tumor-promoting myeloid-derived suppressor cells via oxidative protein folding[J].Journal of immunology,2015,194(4):2004-2010.
[8] 陈风雷.小鼠Zhangfei基因过表达和shRNA干扰慢病毒载体的构建和鉴定[D].杨凌:西北农林科技大学,2013:42-43.
[9] CHEN F L,LIN P F,LI X,et al.Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells[J].Plasmid,2014,76:24-31.
[10] CHEN F L,LI Q,ZHANG J Y,et al.Silencing effect of lentiviral vectors encoding shRNA of Herp on endoplasmic reticulum stress and inflammatory responses in RAW 264.7 macrophages[J].Genetics and molecular research,2015,14(4):17587-17598.
[11] RASCHKE W C,BAIRD S,RALPH P,et al.Functional macrophage cell lines transformed by Abelson leukemia virus[J].Cell,1978,5(1):261-267.
[12] BLESCH A.Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer[J].Methods,2004,33(2):164-172.