大鼠NeuroD2基因的克隆、真核表达载体的构建及其在小鼠MSCs细胞中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2016-1087

生物技术通报 ›› 2017, Vol. 33 ›› Issue (6): 134-141.doi: 10.13560/j.cnki.biotech.bull.1985.2016-1087

大鼠NeuroD2基因的克隆、真核表达载体的构建及其在小鼠MSCs细胞中的表达

姬菩忠^1,2, 赵兴绪¹, 秦文², 宋学文², 张勇¹, 赵红斌^1,2

1. 甘肃农业大学生命科学技术学院,兰州 730070;
2. 兰州军区兰州总医院骨科研究所,兰州 730050

收稿日期:2016-11-29 出版日期:2017-06-26 发布日期:2017-06-19
作者简介:姬菩忠,男,硕士研究生,研究方向：生物化学与分子生物学;E-mail：jipuzhong@163.com
基金资助:
国家自然科学基金面上项目（81470087）,全军十二五面上项目（CWS11C228）

Cloning and Construction of Eukaryotic Expression Vector for NeuroD2 in Rat,and Its Expression in Mouse MSCs

JI Pu-zhong^1,2, ZHAO Xing-xu¹, QIN Wen², SONG Xue-wen², ZHANG Yong¹, ZHAO Hong-bin^1,2

1. College of Life Sciences,Gansu Agricultural University,Lanzhou 730070;
2. Institute of Bone Injury,Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA,Lanzhou 730050

Received:2016-11-29 Published:2017-06-26 Online:2017-06-19

摘要/Abstract

摘要： 克隆大鼠NeuroD2基因,旨在构建pIRES2-AcGFP1-ND2真核表达载体并检测其在小鼠MSCs中的表达。采用RT-PCR技术克隆大鼠NeuroD2基因,分析该蛋白质结构、功能域、细胞定位及与其他物种同源性;构建pIRES2-AcGFP1-ND2表达载体并转染小鼠骨髓间充质干细胞（MSCs）;采用Real-time PCR、免疫荧光及Western blot技术检测重组质粒在小鼠MSCs中的表达。结果表明,大鼠NeuroD2基因CDS区全长1 149 bp,共编码382个氨基酸,属于bHLH家族,二级结构以α-螺旋和无规卷曲为主,空间结构呈现近似“螺旋-环-螺旋（HLH）”结构,主要分布在细胞核,氨基酸序列与家鼠（99%）、罗猴（98%）、人（97.7%）同源性较高;Real-time PCR结果表明转染组NeuroD2基因表达量显著高于对照组（P<0.05）;免疫荧光及Western blot结果显示转染组NeuroD2蛋白表达量显著高于对照组（P<0.05）。成功克隆大鼠NeuroD2基因并构建了pIRES2-AcGFP1-ND2真核表达载体。

关键词: NeuroD2, 基因克隆, 生物信息学分析, pIRES2-AcGFP1-ND2

Abstract: This work aims to clone NeuroD2 gene,construct the eukaryotic expression vector pIRES2-AcGFP1-ND2,and analyze its expression in mouse MSCs. The rat NeuroD2 gene was cloned by RT-PCR,and the protein structure,functional domain,cell localization and homology with other species were analyzed. The constructed pIRES2-AcGFP1-ND2 expression vector was transfected to mouse bone marrow mesenchymal stem cells（MSCs）. The expression of the recombinant plasmid in the mouse MSCs was detected by real time-PCR,Western blot and immunofluorescence. The results showed that,the length of CDS in rat NeuroD2 gene was 1149 bp,and it encoded 382 amino acids,belonging to bHLH family. The secondary structure mainly composed of the α-helices and random coil. Tertiary structure of NeuroD2 was a “helix-loop-helix” structure and mainly localized in nucleus. The NeuroD2 protein of the experimental rat（Rattus norvegicus）shared 99%,98% and 97.7% homology with Mus musculus,Macaca mulatta and Homo sapiens,respectively. The results of real-time PCR,Western blot and immunofluorescence indicated that the expression levels of NueorD2 mRNA and protein in transfected group were significantly higher than that in control group（P < 0.05）. In conclusion,the eukaryotic expression vector pIRES2-AcGFP1-ND2 was constructed successfully.

Key words: NeuroD2, gene cloning, bioinformatics, pIRES2-AcGFP1-ND2

姬菩忠, 赵兴绪, 秦文, 宋学文, 张勇, 赵红斌. 大鼠NeuroD2基因的克隆、真核表达载体的构建及其在小鼠MSCs细胞中的表达[J]. 生物技术通报, 2017, 33(6): 134-141.

JI Pu-zhong, ZHAO Xing-xu, QIN Wen, SONG Xue-wen, ZHANG Yong, ZHAO Hong-bin. Cloning and Construction of Eukaryotic Expression Vector for NeuroD2 in Rat,and Its Expression in Mouse MSCs[J]. Biotechnology Bulletin, 2017, 33(6): 134-141.

参考文献

[1] Bormuth I, Yan K, Yonemasu T, et al. Neuronal basic helix-loop-helix proteins Neurod2/6 regulate cortical commissure formation before midline interactions[J]. Neurosci, 2013, 33（2）：641-651.
[2] Chen F, Moran JT, Zhang Y, et al. The transcription factor NeuroD2 coordinates synaptic innervation and cell intrinsic properties to control excitability of cortical pyramidal neurons[J]. J Physiol, 2016, 594（13）：3729-3744.
[3] Vetrovoi OV, Rybnikova EA, Glushchenko TS, et al. Effects of hypobaric hypoxia in various modes on expression of neurogenesis marker NeuroD2 in the dentate gyrus of rats hippocampus[J]. Bull Exp Biol Med, 2016, 160（4）：510-513.
[4] Fong AP, Yao Z, Zhong JW, et al. Conversion of MyoD to a neurogenic factor：binding site specificity determines lineage[J]. Cell Rep, 2015, 10（12）：1937-1946.
[5] Schwab MHA, Bartholomae B, Heimrich D, et al. Neuronal basic helix-loop-helix proteins（NEX and BETA2/Neuro D）regulate terminal granule cell differentiation in the hip-pocampus[J]. Neurosci, 2011, 20（3）：3714-3724.
[6] Farah MH, Olson JM, Sucic H, et al. Generation of neurons by transient expression of neural bHLH proteins in mammalian cells[J]. Development, 2000, 127（35）：693-702.
[7] 王勇, 陈克平, 姚勤, 等. bHLH 转录因子家族研究进展[J]. 遗传学, 2008, 30（7）：821-830.
[8] Goossens J, Mertens J, Goossens A, et al. Role and functioning of bHLH transcription factors in jasmonate signaling[J]. J Exp Bot, 2016, 7, pii：erw440.
[9] Timothy J, Cherry L, Sui Wang, et al. NeuroD Factors Regulate Cell Fate and Neurite Stratification in the Developing Retina[J]. Neurosci, 2011, 31（20）：7365-7379.
[10] Atchley WR, Fitch WM. A natural classification of the basic helix-loop-helix class of transcription factors[J]. Proc Natl Acad Sci USA, 1998, 94（10）：5172-5176.
[11] Gutierrez MA, Davis SS, Rosko A, et al. A novel AhR ligand, 2AI, protects the retina from environmental stress[J]. Sci Rep, 2016, 6：29025.
[12] Lai HC, Klisch TJ, Roberts R, et al. In vivo neuronal subtype-specific targets of Atoh1（Math1）in dorsal spinal cord[J]. J Neurosci, 2011, 31（30）：10859-10871.
[13] Shahmoradi A, Radyushkin K, Rossner MJ. Enhanced memory consolidation in mice lacking the circadian modulators Sharp1 and -2 caused by elevated Igf2 signaling in the cortex[J]. Proc Natl Acad Sci U S A, 2015, 112（27）：E3582- 3589.
[14] Ma YC, Song MR, Park JP, et al. Regulation of motor neuron specification by phosphorylation of neurogenin 2[J]. Neuron, 2008, 58（4）：65-77.
[15] Bormuth I, Yan K, Yonemasu T, et al. Neuronal basic helix-loop-helix proteins Neurod2/6 regulate cortical commissure formation before midline interactions[J]. J Neurosci, 2013, 33（2）：641-651.
[16] Ravanpay AC, Hansen SJ, Olson JM. Transcriptional inhibition of REST by NeuroD2 during neuronal differentiation[J]. Molecular and Cellular Neurosciences, 2010, 44（2）：178-189.
[17] Qian JY, ChoppM, Liu Z. Mesenchymal stromal cells promote axonal outgrowth alone and synergistically with astrocytes via tPA[J]. PLoS One, 2016, 11：e0168345.
[18] Nakano N, Nakai Y, Seo T-B, et al. Effects of bone marrow stromal cell transplantation through CSF on the Subacute and chronicSpinal cord injury in rats[J]. PLoS One, 2013, 8（9）：e73494.
[19] Lin CH, Stoeck J, Ravanpay AC, et al. Regulation of neuroD2 expression in mouse brain[J]. Developmental Biology, 2004, 265：234-245.
[20] 张全伟, 赵兴绪, 赵红斌等. 红景天苷体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞[J]. 中国组织工程研究, 2012, 16（14）：2496-2503.

大鼠NeuroD2基因的克隆、真核表达载体的构建及其在小鼠MSCs细胞中的表达

Cloning and Construction of Eukaryotic Expression Vector for NeuroD2 in Rat,and Its Expression in Mouse MSCs

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