Rab7表达载体的构建及功能鉴定
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摘要
目的:构建人Rab7与增强型绿色荧光蛋白(EGFP)融合表达载体,研究其对黑素代谢的影响。方法:分离人外周血淋巴细胞,提取细胞总RNA,逆转录成cDNA,以特异性引物扩增Rab7片段,酶切后连接至EGFP-C1载体并转化至感受态大肠杆菌中,测序分析以确定表达载体构建正确。将构建成功的重组质粒转染A375细胞,检测细胞黑素合成酶的表达水平和黑素含量的变化情况。结果:EGFP-Rab7重组质粒经测序鉴定构建正确。重组质粒转染A375细胞后,细胞中的酪氨酸酶(TYR)和酪氨酸酶相关蛋白1(TYPR1)表达水平和细胞黑素含量高于空载质粒组。结论:本研究成功构建了EGFP-Rab7融合表达载体,其可增加细胞中的黑素合成酶表达水平和细胞黑素含量。
Objective: To construct EGFP-Rab7 fusion expression vector, and to investigate its effect on melanin synthesis in A375 cells. Methods: Total RNA was extracted from peripheral lymphocytes for reverse transcription of cDNA. Polychain reaction(PCR) was performed with specific primers to amplify Rab7 fragment. PCR products were digested and inserted into EGFP-C1 vector. The recombinant plasmids EGFP-Rab7 were transformed into E. coli DH5 a. Positive clones were screened and identified by DNA sequencing. The recombinant plasmid was transfected into A375 cells and the expression level of melanogenic enzymes and melanin content was detected. Results: EGFP-Rab7 expression vectors were verified by DNA sequencing. The expression level of tyrosinase and tyrosinase-related protein 1 and melanin content in A375 cells transfected by EGFP-Rab7 was higher than that in empty transfer plasmid group. Conclusion: EGFP-Rab7 fusion expression vector is successfully constructed which can increase the level of melanin and and melanin synthetase.
Objective: To construct EGFP-Rab7 fusion expression vector, and to investigate its effect on melanin synthesis in A375 cells. Methods: Total RNA was extracted from peripheral lymphocytes for reverse transcription of cDNA. Polychain reaction(PCR) was performed with specific primers to amplify Rab7 fragment. PCR products were digested and inserted into EGFP-C1 vector. The recombinant plasmids EGFP-Rab7 were transformed into E. coli DH5 a. Positive clones were screened and identified by DNA sequencing. The recombinant plasmid was transfected into A375 cells and the expression level of melanogenic enzymes and melanin content was detected. Results: EGFP-Rab7 expression vectors were verified by DNA sequencing. The expression level of tyrosinase and tyrosinase-related protein 1 and melanin content in A375 cells transfected by EGFP-Rab7 was higher than that in empty transfer plasmid group. Conclusion: EGFP-Rab7 fusion expression vector is successfully constructed which can increase the level of melanin and and melanin synthetase.
引文
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[8] 谢双德,魏珊珊,张华年,等.mTORC1信号通路在狼疮鼠体内的表达及意义简[J].中国麻风皮肤病杂志,2018,34(8):449-451.
[9] Langemeyer L,Fr?hlich F,Ungermann C.Rab GTPase function in endosome and lysosome biogenesis[J].Trends Cell Biol,2018,28(11):957-970.
[10] Barr F,Lambright DG.Rab GEFs and GAPs[J].Curr Opin Cell Biol,2010,22(4):461-470.
[11] Fukuda M.TBC proteins:GAPs for mammalian small GTPase Rab?[J].Biosci Rep,2011,31(3):159-168.
[12] Ceresa BP,Bahr SJ.rab7 activity affects epidermal growth factor:Epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome[J].J Biol Chem,2006,281(2):1099-1106.
[13] Calvo A,Xiao NQ,Kang J,et al.Alterations in gene expression profiles during prostate cancer progression:Functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors[J].Cancer Res,2002,62(18):5325-5335.
[14] Hida T,Sohma H,Kokai Y,et al.Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes[J].J Dermatol,2011,38(5):432-441.
[15] Marks MS,Heijnen HFG,Raposo G.Lysosome-related organelles:unusual compartments become mainstream[J].Curr Opin Cell Biol,2013,25(4):495-505.
[2] Pfeffer SR.Rab GTPases:master regulators that establish the secretory and endocytic pathways[J].Mol Biol Cell,2017,28(6):712-715.
[3] Kummel D,Ungermann C.Principles of membrane tethering and fusion in endosome and lysosome biogenesis[J].Curr Opin Cell Biol,2014,29:61-66.
[4] Kim JJ,Lipatova Z,Majumdar U,et al.Regulation of golgi cisternal progression by Ypt/Rab GTPases[J].Dev Cell,2016,36(4):440-452.
[5] Srikanth S,Woo JS,Gwack Y.A large Rab GTPase family in a small GTPase world[J].Small GTPases,2017,8(1):43-48.
[6] Tamura K,Ohbayashi N,Maruta Y,et al.Varp is a novel Rab32/38-binding protein that regulates tyrp1 trafficking in melanocytes[J].Mol Biol Cell,2009,20(12):2900-2908.
[7] 王勇,韩建文,白云花,等.内蒙古汉族寻常型银屑病REL基因多态位点检测[J].中国麻风皮肤病杂志,2016,32(6):342-345.
[8] 谢双德,魏珊珊,张华年,等.mTORC1信号通路在狼疮鼠体内的表达及意义简[J].中国麻风皮肤病杂志,2018,34(8):449-451.
[9] Langemeyer L,Fr?hlich F,Ungermann C.Rab GTPase function in endosome and lysosome biogenesis[J].Trends Cell Biol,2018,28(11):957-970.
[10] Barr F,Lambright DG.Rab GEFs and GAPs[J].Curr Opin Cell Biol,2010,22(4):461-470.
[11] Fukuda M.TBC proteins:GAPs for mammalian small GTPase Rab?[J].Biosci Rep,2011,31(3):159-168.
[12] Ceresa BP,Bahr SJ.rab7 activity affects epidermal growth factor:Epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome[J].J Biol Chem,2006,281(2):1099-1106.
[13] Calvo A,Xiao NQ,Kang J,et al.Alterations in gene expression profiles during prostate cancer progression:Functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors[J].Cancer Res,2002,62(18):5325-5335.
[14] Hida T,Sohma H,Kokai Y,et al.Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes[J].J Dermatol,2011,38(5):432-441.
[15] Marks MS,Heijnen HFG,Raposo G.Lysosome-related organelles:unusual compartments become mainstream[J].Curr Opin Cell Biol,2013,25(4):495-505.