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家兔胆固醇酯转移蛋白基因克隆及其真核表达载体的构建

张 鹏1,2， 王艳丽3，  郭世文1， 刘恩岐3   

  1. 1.  西安交通大学医学院第一附属医院神经外科，陕西 西安 710061；2.  西安医学院临床医学院，陕西 西安 710021；3.  西安交通大学医学院实验动物中心，陕西 西安 710061
  • 收稿日期:2012-12-14 出版日期:2013-07-28 发布日期:2013-08-17
  • 通讯作者: 刘恩岐（Tel: 029-82657057，E-mail: liuenqi@mail.xjtu.edu.cn） E-mail:liuenqi@mail.xjtu.edu.cn
  • 作者简介:张 鹏（1976-），男，陕西省高陵县人，讲师，主治医师，在读医学硕士，主要从事神经外科疾病的基础研究。 
  • 基金资助:

     国家自然科学基金资助课题（81070250）

Cloning of rabbit cholesteryl ester transfer protein gene andconstruction of its  eukaryotic expression vector

ZHANG Peng 1,2,WANG Yan-li 3,GUO Shi-wen1,LIU En-qi3   

  1. 1. Department of Neurosurgery,First Affiliated Hospital，College of Medical Sciences,Xi’an Jiaotong University，Xi’an 710061,China；2. School of Clinical Medicine，Xi’an Medical College,Xi’an 710021,China；3. Laboratory Animal Center, College of Medical Sciences,Xi’an
     Jiaotong University，Xi’an 710061,China
  • Received:2012-12-14 Online:2013-07-28 Published:2013-08-17

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摘要：

目的: 克隆家兔胆固醇酯转移蛋白(CETP) 基因，构建其真核表达载体，研究CETP基因的结构与功能，为进一步研究CETP在动脉粥样硬化(As)中的作用机制提供参考。方法: 采用TRIzol提取家兔肝组织总RNA，采用RT-PCR 方法将RNA逆转录为cDNA，以cDNA为模板，用LA Taq酶进行PCR扩增，克隆出CETP基因，将CETP cDNA片段克隆至pMD19-T载体上，对重组的CETP- pMD19-T测序后，登录GenBank进行序列比对，利用nnPredict软件分析预测CETP的二级结构。将CETP
 cDNA片段克隆至pcDNA3.1真核表达载体上，对重组质粒进行PCR及酶切鉴定。结果：测序及序列比对显示与M27486基因序列98%相符；家兔CETP基因cDNA全长1 659 bp，编码序列(CDs区)长1 494 bp，编码498个氨基酸。CETP的二级结构以α螺旋结构为主，相对分子质量为142119.4， pI为4.91。结论: 成功克隆了家兔CETP基因，并成功构建了其真核表达载体，明确了CETP的二级结构。 

关键词:  , 胆固醇酯转移蛋白, 克隆, 序列分析, 真核表达载体

Abstract:

Objective To clone cholesteryl ester transfer protein (CETP) gene of rabbits and to construct its eukaryotic expression vector,and to study the st
ructure and function of CETP gene,and to lay the foundation for further study on the action mechanism of CETP in atherosclerosis(AS).
Methods Total RNA of rabbit liver tissue  was extracted by  TRIzol.RNA was  reversely transcripted into  cDNA by RT-PCR method.Then the CETP gene was cloned by PCR amplification with LA Taq enzyme and the cDNA was regared as template.The CETP cDNA fragment was
cloned into the pMD19-T vector.The recombinant CETP-pMD19-T was sequenced and alignmented with GenBank sequence.The sec
ondary structure of CETP was analyzed by nnPredict software.Then the CETP cDNA fragment was cloned into the pcDNA3.1 eukaryotic expression vector.And the recombinant plasmid was identified by restriction enzyme and PCR.Results The  sequencing and matching  results showed that  it w
as 98%  corresponded to M27486 gene.The full length of CETP cDNA was 1 659 bp,and the length of coded sequence(CDs zone) was 1
 494 bp,and it encoded 498 amino acids. The secondary structure of CETP was the α-helix structure mainly,its molecular weight was 142 119.4，and pI was 4.91.Conclusion The CETP cDNA is successfully cloned，and the CETP eukaryotic expression vector is constructed and the secondary structure of CETP is forecasted  successfully.

Key words: cholesteryl ester transfer protein, cloning, sequence analysis, eukaryotic expression vector

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