MPST基因慢病毒载体的构建及在SH-SY5Y细胞中的表达
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摘要
为构建MPST基因慢病毒表达载体,获得稳定表达外源MPST基因的SH-SY5Y细胞株,本研究通过PCR扩增出MPST目的基因,将其克隆到慢病毒表达载体p EB-GFP (T2A) PURO上,将重组慢病毒表达载体和慢病毒包装质粒系统(pLP/VSVG, pLP1, p LP2)共转染293T细胞,用获得重组的慢病毒液感染SH-SY5Y细胞,嘌呤霉素筛选稳定表达MPST基因的(SH-MPST)细胞株。采用Real-time PCR和Western blotting以及ELISA等方法对筛出来的SH-MPST中的MPST的表达及功能进行鉴定。与空转染组(SH-PEB)相比,SH-MPST细胞中MPST mRNA及蛋白的表达水平显著增加,且细胞内MPST酶活性、酶含量及细胞释放硫化氢的水平均显著增加(p<0.05)。以上研究表明,MPST基因慢病毒表达载体成功构建,并获得稳定表达外源MPST基因的SH-SY5Y细胞株,这将为MPST功能的深入研究提供依据。
In order to construct lentiviral vector of MPST gene and obtain stable overexpression of the exogenous gene in SH-SY5 Y cell line, MPST gene was amplified by polymerase chain reaction(PCR) and then cloned into lentiviral vector pEB-GFP(T2 A) PURO. The 293 T cells were co-transfected with the recombinant lentiviral vector and the lentiviral packaging plasmid system(pLP/VSVG, p LP1, p LP2), then the supernatant of recombinant lentivirus was used to infect SH-SY5 Y cells. Puromycin was used to screen(SH-MPST) the cell line stably expressing the MPST gene. MPST gene overexpressed vector was identified by Real-time PCR, Western blotting and ELISA in the new cell lines. The results showed that compared with SH-PEB group, the expression of MPST m RNA and protein increased significantly, and the MPST activity, enzyme content and the level of released hydrogen sulfide were significantly increased in SH-MPST cells(p<0.05). The above studies indicated that the lentiviral vector carrying MPST and the SH-SY5 Y cell line stably expressing exogenous gene were successfully constructed which will provide the basis for the further study of the function of MPST.
In order to construct lentiviral vector of MPST gene and obtain stable overexpression of the exogenous gene in SH-SY5 Y cell line, MPST gene was amplified by polymerase chain reaction(PCR) and then cloned into lentiviral vector pEB-GFP(T2 A) PURO. The 293 T cells were co-transfected with the recombinant lentiviral vector and the lentiviral packaging plasmid system(pLP/VSVG, p LP1, p LP2), then the supernatant of recombinant lentivirus was used to infect SH-SY5 Y cells. Puromycin was used to screen(SH-MPST) the cell line stably expressing the MPST gene. MPST gene overexpressed vector was identified by Real-time PCR, Western blotting and ELISA in the new cell lines. The results showed that compared with SH-PEB group, the expression of MPST m RNA and protein increased significantly, and the MPST activity, enzyme content and the level of released hydrogen sulfide were significantly increased in SH-MPST cells(p<0.05). The above studies indicated that the lentiviral vector carrying MPST and the SH-SY5 Y cell line stably expressing exogenous gene were successfully constructed which will provide the basis for the further study of the function of MPST.
引文
Billaut-Laden I.,Rat E.,Allorge D.,Crunelle-Thibaut A.,Cauffiez C.,Chevalier D.,Lo-Guidice J.M.,and Broly F.,2006,Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase(MPST),a cyanide detox ification enzyme,Toxicol.Lett.,165(2):101-111
Kashfi K.,2018,The role of hydrogen sulfide in health and disease,Biochem.Pharmacol.,149:1-4
Kimura H.,2011,Hydrogen sulfide:its production and functions,Exp.Physiol.,96(9):833-835
Kimura H.,2013,Physiological role of hydrogen sulfide and polysulfide in the central nervous system,Neurochem.Int.,63(5):492-497
Krishna A.,Biryukov M.,Trefois C.,Antony P.M.,Hussong R.,Lin J.,Hein覿niemi M.,Glusman G.,K觟glsberger S.,Boyd O.,van den Berg B.H.,Linke D.,Huang D.,Wang K.,Hood L.,Tholey A.,Schneider R.,Galas D.J.,Balling R.,and May P.,2014,Systems genomics evaluation of the SH-SY5Yneuroblastoma cell line as a model for Parkinson's disease,BMC Genomics,15(1):1154
Liu Y.P.,and Berkhout B.,2014,HIV-1-based lentiviral vectors,Methods Mol.Biol.,1087:273-284
Moore P.K.,and Whiteman M.,2015,Chemistry,biochemistry and pharmacology of hydrogen sulfide,Handbook of Experimental Pharmacology,230:9
Nagahara N.,2018,Multiple role of 3-mercaptopyruvate sulfurtransferase:antioxidative function,H2S and polysulfide production and possible SOx production,Br.J.Pharmacol.,175(4):577-589
Nagahara N.,Nagano M.,Ito T.,and Suzuki H.,2015,Redox regulation of mammalian 3-mercaptopyruvate sulfurtransferase,Methods Enzymol.,554:229-254
Nagahara N.,Nagano M.,Ito T.,Shimamura K.,Akimoto T.,and Suzuki H.,2013,Antioxidant enzyme,3-mercaptopyruvate sulfurtransferas e-knockout mice exhibit increased anxiety-like behaviors:a model for human mercaptolactate-cysteine disulfiduria,Sci.Rep.,3:1986
Nie Y.,Wang N.,Xu G.Q.,and Pan J.G.,2016,Effect of sodium arsenite on cell growth and ex-pression of 3MST in SH-SY5Y cells,Zhongguo Yaolixue Tongbao(Chinese Pharmacological Bulletin),32(8):1182-1183(聂杨,王念,徐国强,潘际刚,2016,亚砷酸钠对SH-SY5Y细胞生长和3MST表达的影响,中国药理学通报,32(8):1182-1183)
Papapetropoulos A.,Whiteman M.,and Cirino G.,2015,Pharmacological tools for hydrogen sulphide research:a brief,introductory guide for beginners,Br.J.Pharmacol.,172(6):1633-1637
Shefa U.,Kim M.S.,Jeong N.Y.,and Jung J.,2018,Antioxidant and cell-signaling functions of hydrogen sulfide in the central nervous system,Oxid.Med.Cell.Longev.,1:1873962
Yu F.,Zhao J.,Tang C.H.,and Geng B.,2010,Effect of synthesized GYY4137,a slowly releasing hydrogen sulfide donor,on cell viability and distribution of hydrogen sulfide in mice,J.Peking Uni.(Heal.Sci.),42(5):493-497
Zhao H.,Chan S.J.,Ng Y.K.,and Wong P.T.,2013,Brain3-mercaptopyruvate sulfurtransferase(3MST):cellular localization and downregulation after acute stroke,PLo S One,8(6):e67322
Kashfi K.,2018,The role of hydrogen sulfide in health and disease,Biochem.Pharmacol.,149:1-4
Kimura H.,2011,Hydrogen sulfide:its production and functions,Exp.Physiol.,96(9):833-835
Kimura H.,2013,Physiological role of hydrogen sulfide and polysulfide in the central nervous system,Neurochem.Int.,63(5):492-497
Krishna A.,Biryukov M.,Trefois C.,Antony P.M.,Hussong R.,Lin J.,Hein覿niemi M.,Glusman G.,K觟glsberger S.,Boyd O.,van den Berg B.H.,Linke D.,Huang D.,Wang K.,Hood L.,Tholey A.,Schneider R.,Galas D.J.,Balling R.,and May P.,2014,Systems genomics evaluation of the SH-SY5Yneuroblastoma cell line as a model for Parkinson's disease,BMC Genomics,15(1):1154
Liu Y.P.,and Berkhout B.,2014,HIV-1-based lentiviral vectors,Methods Mol.Biol.,1087:273-284
Moore P.K.,and Whiteman M.,2015,Chemistry,biochemistry and pharmacology of hydrogen sulfide,Handbook of Experimental Pharmacology,230:9
Nagahara N.,2018,Multiple role of 3-mercaptopyruvate sulfurtransferase:antioxidative function,H2S and polysulfide production and possible SOx production,Br.J.Pharmacol.,175(4):577-589
Nagahara N.,Nagano M.,Ito T.,and Suzuki H.,2015,Redox regulation of mammalian 3-mercaptopyruvate sulfurtransferase,Methods Enzymol.,554:229-254
Nagahara N.,Nagano M.,Ito T.,Shimamura K.,Akimoto T.,and Suzuki H.,2013,Antioxidant enzyme,3-mercaptopyruvate sulfurtransferas e-knockout mice exhibit increased anxiety-like behaviors:a model for human mercaptolactate-cysteine disulfiduria,Sci.Rep.,3:1986
Nie Y.,Wang N.,Xu G.Q.,and Pan J.G.,2016,Effect of sodium arsenite on cell growth and ex-pression of 3MST in SH-SY5Y cells,Zhongguo Yaolixue Tongbao(Chinese Pharmacological Bulletin),32(8):1182-1183(聂杨,王念,徐国强,潘际刚,2016,亚砷酸钠对SH-SY5Y细胞生长和3MST表达的影响,中国药理学通报,32(8):1182-1183)
Papapetropoulos A.,Whiteman M.,and Cirino G.,2015,Pharmacological tools for hydrogen sulphide research:a brief,introductory guide for beginners,Br.J.Pharmacol.,172(6):1633-1637
Shefa U.,Kim M.S.,Jeong N.Y.,and Jung J.,2018,Antioxidant and cell-signaling functions of hydrogen sulfide in the central nervous system,Oxid.Med.Cell.Longev.,1:1873962
Yu F.,Zhao J.,Tang C.H.,and Geng B.,2010,Effect of synthesized GYY4137,a slowly releasing hydrogen sulfide donor,on cell viability and distribution of hydrogen sulfide in mice,J.Peking Uni.(Heal.Sci.),42(5):493-497
Zhao H.,Chan S.J.,Ng Y.K.,and Wong P.T.,2013,Brain3-mercaptopyruvate sulfurtransferase(3MST):cellular localization and downregulation after acute stroke,PLo S One,8(6):e67322