pcDNA3.1/myc-His-DJ-1M26I真核表达载体构建及其在NIH3T3细胞中表达

doi:10.3969/j.issn.1674-5817.2012.02.006

实验动物与比较医学 ›› 2012, Vol. 32 ›› Issue (2): 111-115.DOI: 10.3969/j.issn.1674-5817.2012.02.006

pcDNA3.1/myc-His-DJ-1^M26I真核表达载体构建及其在NIH3T3细胞中表达

张梅英¹, 王惟¹, 徐影琪¹, 赵越¹, 杨葳¹, 于萌¹, 郭晓冲¹, 秦英¹, 郑志红^1,2

1.中国医科大学实验动物部辽宁省转基因动物研究重点实验室实验动物生物工程与转基因技术应用重点实验室, 沈阳110001;
2.中国医科大学病理学与病理生理学研究室, 沈阳 110001

收稿日期:2011-07-01 出版日期:2012-04-25 发布日期:2012-04-25
作者简介:张梅英(1965-),女,副教授,长期从事人类疾病转基因动物研究。E-mail:zhmeiying@163.com
基金资助:
辽宁省科技计划项目(编号: 2009408001-1)

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26Iand Expression in NIH3T3 Cells

ZHANG Mei-ying¹, XU Ying-qi¹, WANG Wei¹, ZHAO Yue¹, YANG Wei¹, YU Meng¹, GUO Xiao-chong¹, QIN Ying¹, ZHENG Zhi-hong^1,2

1. Laboratory Animal Center, China Medical University, Shenyang 110001, China;
2. Department of Pathology and Pathophysiology Research, China Medical University, Shenyang 110001, China

Received:2011-07-01 Online:2012-04-25 Published:2012-04-25

摘要/Abstract

摘要： 目的构建pcDNA3.1/myc-his-DJ-1和pcDNA3.1/myc-his-DJ-1^M26I重组表达载体,为进一步研究DJ-1^M26I基因的功能及建立转基因动物模型奠定基础。方法采用突变试剂盒将第26位氨基酸进行突变,分别构建pcDNA3.1/myc-his-DJ-1和pcDNA3.1/myc-his-DJ-1^M26I重组表达载体,采用脂质体转染的方法分别将pcDNA3.1/myc-his-DJ-1和pcDNA3.1/myc-his-DJ-1^M26I质粒转染NIH3T3细胞并用G418压力筛选稳定克隆,对获得的2种转染细胞在DNA水平、转录水平和蛋白质水平鉴定。结果 pcDNA3.1/myc-his-DJ-1和pcDNA3.1/myc-his-DJ-1^M26I质粒转染NIH3T3细胞经G418筛选后,经PCR检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1^M26I基因的NIH3T3阳性细胞组增殖速率低于正常NIH3T3细胞组(P<0.05)。结论成功构建pcDNA3.1/myc-his-DJ-1和pcDNA3.1/myc-his-DJ-1^M26I重组表达质载体。

关键词: DJ-1, NIH3T3细胞, 帕金森氏病

Abstract: Objective To construct the expression vector pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I to lay a foundation of further study on function of DJ-1^M26I and establish transgenic animals. Methods The 26^th amino acid of DJ-1 was mutated by gene mutation kit, then pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I were constructed. The two plasmids were transfected into NIH3T3 cells respectively using liposome method, then the cells were selected with G418 and detected the DJ-1 gene expression at DNA, transcription and protein levels. Results 1 clone of pcDNA3.1/myc-his-DJ-1 and 3 clones of pcDNA3.1/myc-his-DJ-1^M26I were obtained after G418 selection by PCR analysis. The expression of DJ-1-His were detected by RT-PCR and Western blot in pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-his-DJ-1^M26Itransfected cells. The profieration rate of DJ-1^M26I transfected cells was lower than normal NIH3T3 cells (P<0.05) by MTT essay. Conclusion The expression vectors pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I were constructed successfully.

Key words: DJ-1, NIH3T3 cell, Parkinson's disease

中图分类号:

Q95-33

张梅英, 王惟, 徐影琪, 赵越, 杨葳, 于萌, 郭晓冲, 秦英, 郑志红. pcDNA3.1/myc-His-DJ-1^M26I真核表达载体构建及其在NIH3T3细胞中表达[J]. 实验动物与比较医学, 2012, 32(2): 111-115.

ZHANG Mei-ying, XU Ying-qi, WANG Wei, ZHAO Yue, YANG Wei, YU Meng, GUO Xiao-chong, QIN Ying, ZHENG Zhi-hong. Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26Iand Expression in NIH3T3 Cells[J]. Laboratory Animal and Comparative Medicine, 2012, 32(2): 111-115.

[1]	张梅英，蔺美娜，杨葳，董婉维，李兆阳，王惟，郑志红，王禄增. 转染DJ-1和DJ-1^L166P基因的NIH3T3细胞中tau基因表达的研究[J]. , 2008, 28(6): 361-366.
[2]	张梅英, 李华, 董婉维, 杨葳, 秦英, 汪瑛, 周生来, 于洋, 王惟, 王太一, 王禄增. 具有潮霉素B抗性的NIH3T3细胞饲养层的制备[J]. , 2005, 25(3): 131-135.

pcDNA3.1/myc-His-DJ-1^M26I真核表达载体构建及其在NIH3T3细胞中表达

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26Iand Expression in NIH3T3 Cells

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pcDNA3.1/myc-His-DJ-1M26I真核表达载体构建及其在NIH3T3细胞中表达

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1M26I and Expression in NIH3T3 Cells

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pcDNA3.1/myc-His-DJ-1^M26I真核表达载体构建及其在NIH3T3细胞中表达

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26Iand Expression in NIH3T3 Cells