慢病毒载体介导下DJ-1过表达细胞模型的建立
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摘要
目的构建人野生型DJ-1及其L166P突变体的慢病毒载体并探讨慢病毒载体在构建基因过表达细胞模型中的作用。方法分别构建野生型DJ-1与L166P突变型DJ-1慢病毒载体质粒。进行测序确定比对正确后,进行质粒的大量扩增与制备并转染包装细胞系HEK293T细胞,荧光法和Western blot检测野生型DJ-1与L166P突变型DJ-1在细胞系中的表达。在确定目的蛋白正确表达之后,大量转染HEK293T细胞进行包装并生产携带目的基因的慢病毒颗粒。测定病毒上清滴度后感染PC12细胞,荧光显微镜和Western blot观察GFP荧光强度以及目的蛋白的表达,确定病毒的感染效率。结果成功构建携带DJ-1野生型及其突变体的慢病毒载体。该病毒载体可以转染进入HEK293T细胞内且目的蛋白能够正确表达。LV-DJ-1与LV-DJ-1/L166P的病毒滴度分别为2×10~9TU/m L与2×10~8TU/m L。病毒上清可以高效感染PC12细胞,绝大多数细胞可表达目的蛋白。外源野生型DJ-1和L166P突变体的蛋白表达量分别是内源性含量的315%和285%。结论慢病毒感染细胞效率很高,是很好的制备基因过表达细胞的方法。通过慢病毒载体介导,本研究获得了DJ-1及其突变体的过表达细胞模型。该模型可以用于后续DJ-1功能研究。
Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166 P mutant,and to investigate the role of lentiviral vector in gene overexpressing cell model. Methods Wild type DJ-1 and L166 P mutant DJ-1 lentiviral vector plasmids were respectively constructed. After sequencing and comparing correctly,the plasmid was amplified and transfected into HEK293 T cell line. Expression of WT DJ-1 and L166 P mutant DJ-1 in cell lines was detected by fluorescence and Western blot. After determining the accurate expression of the target protein,a large amount of HEK293 T cells was transfected and packaged to produce lentiviral particles.The PC12 cells were infected with the titer of virus supernatant. The fluorescence intensity of GFP and the expression of target protein were observed by fluorescence microscope and Western blot method,and the infection effi-ciency of the virus was determined. Results Lentiviral vectors carrying wild type DJ-1 and its mutants were successfully constructed. The virus vector can be transfected into HEK293 T cells and the target protein can be correctly expressed. The viral titers of LV-DJ-1 and LV-DJ-1/L166 P were 2×10~9 TU/m L and 2×10~8 TU/m L,respectively.Virus supernatant can efficiently infect PC12 cells,and most cells can express target proteins. The protein expressions of exogenous wild-type DJ-1 and L166 P mutants were 315% and 285% of endogenous content,respectively.Conclusions Lentivirus vector can infect cells efficiently,and it is a good way to prepare gene over expressing cell model. A cell model overexpressing DJ-1 or its L166 P mutant is successfully prepared. The model can be used for subsequent DJ-1 function research.
Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166 P mutant,and to investigate the role of lentiviral vector in gene overexpressing cell model. Methods Wild type DJ-1 and L166 P mutant DJ-1 lentiviral vector plasmids were respectively constructed. After sequencing and comparing correctly,the plasmid was amplified and transfected into HEK293 T cell line. Expression of WT DJ-1 and L166 P mutant DJ-1 in cell lines was detected by fluorescence and Western blot. After determining the accurate expression of the target protein,a large amount of HEK293 T cells was transfected and packaged to produce lentiviral particles.The PC12 cells were infected with the titer of virus supernatant. The fluorescence intensity of GFP and the expression of target protein were observed by fluorescence microscope and Western blot method,and the infection effi-ciency of the virus was determined. Results Lentiviral vectors carrying wild type DJ-1 and its mutants were successfully constructed. The virus vector can be transfected into HEK293 T cells and the target protein can be correctly expressed. The viral titers of LV-DJ-1 and LV-DJ-1/L166 P were 2×10~9 TU/m L and 2×10~8 TU/m L,respectively.Virus supernatant can efficiently infect PC12 cells,and most cells can express target proteins. The protein expressions of exogenous wild-type DJ-1 and L166 P mutants were 315% and 285% of endogenous content,respectively.Conclusions Lentivirus vector can infect cells efficiently,and it is a good way to prepare gene over expressing cell model. A cell model overexpressing DJ-1 or its L166 P mutant is successfully prepared. The model can be used for subsequent DJ-1 function research.
引文
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[2]Gu L,Cui T,Fan C,et al.Involvement of ERK1/2 signalingpathway in DJ-1-induced neuroprotection againstoxidative stres[J].Biochem Biophys Res Commun,2009,383:74-469.
[3]Honbou K,Suzuki NN,Horiuchi M,et al.The crystal structure of DJ-1,a protein related to male fertility and Parkinson's disease[J].J Biol Chem,2003,278:3-17.
[4]Guo M.What have we learned from Drosophila models of Parkinson's disease[J].Prog Brain Res,2010;184:3-16.
[5]Ghazavi F,Fazlali Z,Banihosseini SS,et al.PRKN,DJ-1,and PINK1 screening identifies novel splice site mutation in PRKN and two novel DJ-1mutations[J].Mov Disord,2011,26:9-80.
[6]Schapira AH,Hartley A,Cleeter MW.Free radicals and mitochondrial dysfunction in Parkinson's disease[J].Biochem Soc Trans,1993,21:367-370.
[7]Reiser J.Production and concentration of pseudotyped HIV-1-based gene transfer vectors[J].Gene Ther,2000,7:3-910.
[8]Liu J,Jones KL,Sumer H,et al.Stable transgene expression in human embryonic stem cells after simple chemical transfeefion[J].Mol Reprod Dev,2009,76:580-586.
[9]Nakagawa T,Hoogenraad CC.Lentiviral transgenesis[J].Methods Mol Biol,2009,530:391-405.
[10]Fassler R.Lentiviral transgene vectors[J].EMBO Rep,2004,5:28-29.
[11]Tiscornia G,Singer O,Verma IM.Production and purification of lentiviral vectors[J].Nat Protoc,2006;1:5-241.
[12]Zhao J,Zhi X,Pan L,et al.Trehalose inhibits A53T mutantα-synuclein overexpression and neurotoxicity in transduced PC12 cells[J].Molecules,2017,22:8-9.
[13]Subramaniam SR,Chesselet MF.Mitochondrial dysfunction and oxidative stress in Parkinson's disease[J].Prog Neurobiol,2013,106-107:17-32.
[14]Le W,Pan T,Huang M,et al.Decreased NURR1 gene expression in patients with Parkinson's disease[J].J Neurol Sci,2008,273:29-33.