表达载脂蛋白L1稳定细胞系的构建
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摘要
目的利用Cumate系统实现APOL1基因在293T细胞内的可诱导稳定表达。方法通过查询NCBI数据库,设计APOL1基因编码区全长引物,克隆至Cumate慢病毒质粒。嘌呤霉素筛选包装慢病毒感染的293T细胞,阳性细胞扩大培养后用不同浓度的Cumate诱导表达,利用Real-time PCR和Western blot验证APOL1的表达情况。结果构建的293T细胞系可被Cumate诱导表达APOL1,且不同浓度的Cumate对细胞表达载脂蛋白L1有不同的影响。Real-time PCR和Western blot均证明在诱导48 h后,随着诱导剂浓度的增加,APOL1表达增多,在Cumate为50μg/ml时APOL1达到最高值。结论成功构建了可诱导表达APOL1基因的293T细胞系,为研究APOL1在EV71感染人体细胞过程中的作用机制提供了稳定的实验材料。
Objective To construct a 293 T cell line with inducible expression of the APOL1 gene by a cumate-inducible expression system. Methods Primers for the APOL1 coding sequence were designed by referring to the NCBI database. APOL1 was cloned into a cumate lentivirus plasmid. 293 T cells were infected with the packaged lentivirus and screened using puromycin. Expression was induced in cells via a serial concentration of cumate. The level of expression of the APOL1 gene was detected using real-time PCR and Western blotting. Results A 293 T cell line with inducible expression of APOL1 was successfully constructed. The level of expression is related to the concentration of cumate. Both real-time PCR and Western blotting confirm that the level of APOL1 expression increases with an increase in the concentration of cumate. Peak expression occurs at a concentration of 50 μg/ml. The level of expression is about 10-fold compared to the concentration at 0 μg/ml(F=537.9,P<0.01). Conclusion A 293 T cell line with inducible expression of the APOL1 gene was successfully constructed. This cell line can help with further research into the mechanism by which enterovirus A71 infects human cells.
Objective To construct a 293 T cell line with inducible expression of the APOL1 gene by a cumate-inducible expression system. Methods Primers for the APOL1 coding sequence were designed by referring to the NCBI database. APOL1 was cloned into a cumate lentivirus plasmid. 293 T cells were infected with the packaged lentivirus and screened using puromycin. Expression was induced in cells via a serial concentration of cumate. The level of expression of the APOL1 gene was detected using real-time PCR and Western blotting. Results A 293 T cell line with inducible expression of APOL1 was successfully constructed. The level of expression is related to the concentration of cumate. Both real-time PCR and Western blotting confirm that the level of APOL1 expression increases with an increase in the concentration of cumate. Peak expression occurs at a concentration of 50 μg/ml. The level of expression is about 10-fold compared to the concentration at 0 μg/ml(F=537.9,P<0.01). Conclusion A 293 T cell line with inducible expression of the APOL1 gene was successfully constructed. This cell line can help with further research into the mechanism by which enterovirus A71 infects human cells.
引文
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[12] Monajemi H,Fontijn RD,Pannekoek H,et al.The apolipoprotein L gene cluster has emerged recently in evolution and is expressed in human vascular tissue[J].Genomics,2002,79(4):539-46.
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[14] Park TS,Kim SW,Lee JH.Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells[J].Asian-Australas J Anim Sci,2017,30(6):886-92.
[15] Poulain A,Perret S,Malenfant F,et al.Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch[J].J Biotechnol,2017(255):16-27.
[16] Li FJ,Xu ZS,Aye HM,et al.An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei[J].Mol Biochem Parasitol,2017(214):101-4.
[2] Vanhamme L,Paturiaux-Hanocq F,Poelvoorde P,et al.Apolipoprotein L-I is the trypanosome lytic factor of human serum[J].Nature,2003,422(6927):83-7.
[3] Taylor HE,Khatua AK,Popik W.The innate immune factor apolipoprotein L1 restricts HIV-1 infection[J].J Virol,2014,88(1):592-603.
[4] Deng L,Jia HL,Liu CW,et al.Proteomic analysis of extremely severe hand,foot and mouth disease infected by enterovirus 71[J].BMC Infect Dis,2013(13):383.
[5] Page NM,Butlin DJ,Lomthaisong K,et al.The human apolipoprotein L gene cluster:identification,classification,and sites of distribution[J].Genomics,2001,74(1):71-8.
[6] Wan G,Zhaorigetu S,Liu Z,et al.Apolipoprotein L1,a novel Bcl-2 homology domain 3-only lipid-binding protein,induces autophagic cell death[J].J Biol Chem,2008,283(31):21540-9.
[7] Lan X,Jhaveri A,Cheng K,et al.APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability[J].Am J Physiol Renal Physiol,2014,307(3):326-6.
[8] Nichols B,Jog P,Lee J H,et al.Innate immunity pathways regulate the nephropathy gene Apolipoprotein L1[J].Kidney Int,2015,87(2):332-42.
[9] Cheng D,Weckerle A,Yu Y,et al.Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells[J].J Lipid Res,2015,56(8):1583-93.
[10] Khatua AK,Cheatham AM,Kruzel ED,et al.Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1[J].Am J Physiol Cell Physiol,2015,309(1):22-37.
[11] Olabisi OA,Zhang JY,Verplank L,et al.APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases[J].Proc Natl Acad Sci USA,2016,113(4):830-7.
[12] Monajemi H,Fontijn RD,Pannekoek H,et al.The apolipoprotein L gene cluster has emerged recently in evolution and is expressed in human vascular tissue[J].Genomics,2002,79(4):539-46.
[13] Zhaorigetu S,Wan G,Kaini R,et al.ApoL1,a BH3-only lipid-binding protein,induces autophagic cell death[J].Autophagy,2008,4(8):1079-82.
[14] Park TS,Kim SW,Lee JH.Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells[J].Asian-Australas J Anim Sci,2017,30(6):886-92.
[15] Poulain A,Perret S,Malenfant F,et al.Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch[J].J Biotechnol,2017(255):16-27.
[16] Li FJ,Xu ZS,Aye HM,et al.An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei[J].Mol Biochem Parasitol,2017(214):101-4.