中华绒螯蟹金属硫蛋白基因在A549细胞中的表达及鉴定
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摘要
目的:克隆中华绒螯蟹金属硫蛋白(metallothionein,MT)基因至真核表达载体,表达蛋白,为进一步研究中华绒螯蟹金属硫蛋白的结构和功能打下基础。方法:设计特异性引物,PCR扩增得到MT基因编码序列,定向克隆至带GFP标签的真核表达载体p EGFP-C1,双酶切及测序鉴定重组质粒后转染A549细胞,荧光观察及Western Blot检测目的蛋白表达情况。结果:通过双酶切和测序确认成功构建了中华绒螯蟹MT基因的真核表达载体;重组质粒转染A549细胞24 h后观察到了绿色荧光,细胞裂解物经过Western Blot检测到符合预期的MT-GFP融合蛋白特异性条带。结论:成功构建了中华绒螯蟹MT基因的真核表达载体,证实了该基因能在A549细胞中成功表达,为进一步研究MT的结构功能打下了基础。
Objective: To clone the metallothionein( MT) gene from Eriocheir sinensis into an eukaryotic expression vector and express the MT proteins,so as to lay a foundation for further study on the structure and function of MT from Eriocheir sinensis. Methods: Specific primers were designed for PCR amplification to obtain the MT gene coding sequence,which was cloned into the GFP-tagged eukaryotic expression vector p EGFP-C1.The recombinant plasmid was validated by double enzyme digestion and sequencing,and then transfected into the A549 cells. Fluorescence microscopy and Western blot was used to examine the expression of target proteins.Results: The eukaryotic expression vector of MT gene of Eriocheir sinensis was successfully constructed by double digestion and sequencing. Green fluorescence was observed after transfection of A549 cells with recombinant plasmid for 24 hours. Western blotting of the cell lysates identified specific band of GFP fusion protein. Conclusion: The eukaryotic expression vector of MT gene of Eriocheir sinensis was successfully constructed,which confirmed that MT gene may successfully express in A549 cells. This study lays a foundation for further study of the structure and function of MT.
Objective: To clone the metallothionein( MT) gene from Eriocheir sinensis into an eukaryotic expression vector and express the MT proteins,so as to lay a foundation for further study on the structure and function of MT from Eriocheir sinensis. Methods: Specific primers were designed for PCR amplification to obtain the MT gene coding sequence,which was cloned into the GFP-tagged eukaryotic expression vector p EGFP-C1.The recombinant plasmid was validated by double enzyme digestion and sequencing,and then transfected into the A549 cells. Fluorescence microscopy and Western blot was used to examine the expression of target proteins.Results: The eukaryotic expression vector of MT gene of Eriocheir sinensis was successfully constructed by double digestion and sequencing. Green fluorescence was observed after transfection of A549 cells with recombinant plasmid for 24 hours. Western blotting of the cell lysates identified specific band of GFP fusion protein. Conclusion: The eukaryotic expression vector of MT gene of Eriocheir sinensis was successfully constructed,which confirmed that MT gene may successfully express in A549 cells. This study lays a foundation for further study of the structure and function of MT.
引文
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[15]Deng X,Qing L I,Sun D,et al. Bioaccumulation of Mercury by Genetically Engineered Escherichia coli from Real Electrolytic Wastewater[J]. Journal of Xiamen University,2000,21(S1):330-333.
[16]Zhao,X. W,Zhou,et al. Simultaneous mercury bioaccumulation and cell propagation by genetically engineered Escherichia coli[J]. Process Biochemistry,2005,40(5):1611-1616.
[17]Krishnaswamy R,Wilson D B. Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II)bioaccumulation[J]. Applied&Environmental Microbiology,2000,66(12):5383-5386.
[18]曹晓敏.重组蟹金属硫蛋白质原核表达、分离纯化及在动物烫伤治疗中的应用[D].广州医学院,2005.
[19]刘进平,何永吉,王兰.河南华溪蟹金属硫蛋白分泌型表达载体的构建、表达及纯化[J].食品科学,2014(17):128-132.
[20]曹晓敏,李冰,陈祯,等.中华绒螯蟹重组金属硫蛋白的原核表达与分离纯化[J].生物医学工程学杂志,2007,24(2):409-412.
[2]Margoshes M,Vallee B L. A CADMIUM PROTEIN FROM EQUINE KIDNEY CORTEX[J]. Journal of the American Chemical Society,1957,79(17):4813-4814.
[3] CarpenèE,Andreani G,Isani G. Metallothionein functions and structural characteristics[J]. Journal of Trace Elements in Medicine and Biology,2007,21:35-39.
[4]Liu Z,Ye Q,Wu L,et al. Metallothionein 1 family profiling identifies MT1X as a tumor suppressor involved in the progression and metastastatic capacity of hepatocellular carcinoma.[J]. Molecular Carcinogenesis,2018.
[5]Leung J Y K,Bennett W R,King A E,et al. The impact of metallothionein-II on microglial response to tumor necrosis factor-alpha(TNFα)and downstream effects on neuronal regeneration[J]. Journal of Neuroinflammation,2018,15(1):56.
[6]Zheng Y,Jiang L,Xu N,et al. Metallothionein 1H functions as a tumor suppressor in hepatocellular carcinoma[J]. Annals of Oncology,2016,27.
[7]Tariba B,ivkovi T,Mariji V F,et al. Does the Serum Metallothionein Level Reflect the Stage of Testicular Germ Cell Tumor[J]. Archives of Medical Research,2016,47(3):232-235.
[8] Cherian M G,Jayasurya A,Bay B H. Metallothioneins in human tumors and potential roles in carcinogenesis[J].Mutation Research/fundamental&Molecular Mechanisms of Mutagenesis,2003,533(1):201-209.
[9]Masters B A,Quaife C J,Erickson J C,et al. Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles.[J]. Journal of Neuroscience,1994,14(10):5844-5857.
[10]Shahpiri A,Mohammadzadeh A. Bioaccumulation of Arsenic by Engineered Escherichia coli Cells Expressing Rice Metallothionein Isoforms[J]. Current Microbiology,2018,75(11):1537-1542.
[11]Su Y J,Lin J Q,Lin J Q,et al. Bioaccumulation of Arsenic in recombinant Escherichia coli expressing human metallothionein[J]. Biotechnology&Bioprocess Engineering,2009,14(5):565-570.
[12]Suleman A,Shakoori A R. Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1(TMCd1)gene in Escherichia coli[J]. Journal of Cellular Biochemistry,2011:n/a-n/a.
[13]Shahpiri A,Mohammadzadeh A. Mercury removal by engineered Escherichia coli cells expressing different rice metallothionein isoforms[J]. Annals of Microbiology,2018,68(3):145-152.
[14] Deng X,Wilson D. Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli[J].Applied Microbiology&Biotechnology,2001,56(1-2):276-279.
[15]Deng X,Qing L I,Sun D,et al. Bioaccumulation of Mercury by Genetically Engineered Escherichia coli from Real Electrolytic Wastewater[J]. Journal of Xiamen University,2000,21(S1):330-333.
[16]Zhao,X. W,Zhou,et al. Simultaneous mercury bioaccumulation and cell propagation by genetically engineered Escherichia coli[J]. Process Biochemistry,2005,40(5):1611-1616.
[17]Krishnaswamy R,Wilson D B. Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II)bioaccumulation[J]. Applied&Environmental Microbiology,2000,66(12):5383-5386.
[18]曹晓敏.重组蟹金属硫蛋白质原核表达、分离纯化及在动物烫伤治疗中的应用[D].广州医学院,2005.
[19]刘进平,何永吉,王兰.河南华溪蟹金属硫蛋白分泌型表达载体的构建、表达及纯化[J].食品科学,2014(17):128-132.
[20]曹晓敏,李冰,陈祯,等.中华绒螯蟹重组金属硫蛋白的原核表达与分离纯化[J].生物医学工程学杂志,2007,24(2):409-412.
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