中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (50): 9437-9440.doi: 10.3969/j.issn.1673-8225.2011.50.031

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

CPT-Ⅱ基因4号外显子质粒构建与变异分析

姚登兵1，姚  敏2，王  理2，董志珍3, 姚登福4   

  1. 1南通大学神经再生重点实验室，江苏省南通市  226001
    2南通大学附属医院医学检验中心，江苏省南通市  226001
    3南通大学医学院诊断学教研室，江苏省南通市 226001
    4南通大学附属医院临床医学研究中心，江苏省南通市  226001
  • 收稿日期:2011-06-01 修回日期:2011-07-16 出版日期:2011-12-10 发布日期:2011-12-10
  • 通讯作者: 姚登福，博士，教授，南通大学附属医院临床医学研究中心，江苏省南通市 226001 yaodf@ahnmc.com
  • 作者简介:姚登兵☆，男，1974年生，医学博士，副教授。 yaodb@ntu.edu.cn 并列第一作者：董志珍，女，1959年生，副研究员。 dongzz@ahnmc.com
  • 基金资助:

    南通大学自然科学基金项目资助(09ZY014，09B16，09ZY007)；江苏省高校自然科学基金项目资助(09KJB310012)；江苏省自然科学基金研究项目(BK2010282)部分资助。

Plasmid construction and variation analysis of exon-4 of carnitine palmitoyltransferase-Ⅱ gene

Yao Deng-bing1, Yao Min2, Wang Li2, Dong Zhi-zhen3, Yao Deng-fu4   

  1. 1Key Laboratory of Neuroregeneration, Nantong University, Nantong  226001, Jiangsu Province, China
    2Center of Medical Examination, Affiliated Hospital of Nantong University, Nantong  226001, Jiangsu Province, China
    3Department of Diagnostics, School of Medicine, Nantong University, Nantong  226001, Jiangsu Province, China
    4Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong  226001, Jiangsu Province, China
  • Received:2011-06-01 Revised:2011-07-16 Online:2011-12-10 Published:2011-12-10
  • Contact: Yao Deng-fu, Doctor, Professor, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China yaodf@ahnmc.com
  • About author:Yao Deng-bing☆, Doctor, Associate professor, Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu Province, China yaodb@ntu.edu.cn Dong Zhi-zhen, Associate researcher, Department of Diagnostics, School of Medicine, Nantong University, Nantong 226001, Jiangsu Province, China dongzz@ahnmc. com Yao Deng-bing and Dong Zhi-zhen contributed equally to this paper.
  • Supported by:

    Natural Science Foundation of Nantong University, No. 09ZY014*,  09B16*, 09ZY007*; Natural Science Foundation for Colleges and Universities in Jiangsu Province, No. 09KJB310012*; Natural Science Foundation of Jiangsu Province, No. BK2010282*

PDF

589

被引次数

1

摘要：

背景：肉毒碱棕榈酰转移酶Ⅱ(Carnitine palmitoyltransferase-Ⅱ, CPT-Ⅱ)位于细胞线粒体内膜上，是脂肪酸氧化过程中起关键作用的酶之一。目前关于流感病毒感染与CPT-Ⅱ基因变异尚未见报道。
目的：构建CPT-Ⅱ第4号外显子(E4)表达质粒及其核苷酸变异分析。
方法：从流感病毒感染2例患者外周血中提取DNA，以PCR法扩增CPT-Ⅱ基因的第4号外显子，将其片段插入pGEM-T载体，以T4 DNA连接酶，构建T-CPT-Ⅱ E4，转化大肠杆菌DH5α细胞，增菌，制备质粒，EcoR I酶切、测序和变异分析。
结果与结论：成功构建重组表达质粒pGEM-T-CPT-ⅡE4，经DNA测序，证实质粒中插入CPT-Ⅱ第4号外显子完整序列，全长1 305个核苷酸，编码435个氨基酸；与Genebank源序列对照比较，发现含有两个变异位点：1 618(GTC→ATC)和1 858 (TTT→TCT)，对应氨基酸为V368I和F448L。

关键词: 肉毒碱棕榈酰转移酶Ⅱ(CPT-Ⅱ), 外显子4, 基因扩增, 变异, 流感病毒A

Abstract:

BACKGROUND: Carnitine palmitoyltransferase-Ⅱ (CPT-Ⅱ) is located in the inner mitochondrial membrane and is one of the pivotal enzymes during the fatty acid oxidation. Currently, there are no studies about influenza virus infection and variation of CPT-Ⅱ gene.
OBJECTIVE: To construct the plasmid expressing exon-4 of CPT-Ⅱ gene (CPT-Ⅱ-E4) and analyze its mutation.
METHODS: Human CPT-Ⅱ DNA from the blood of two patients infected with influenza virus was involved. After the PCR amplification, the product of CPT-Ⅱ-E4 was cloned into pGEMD-T vector. The T-CPT-ⅡE4 was transferred to E. coli DH5α cells after prepared with T4 DNA higase. Then propagation of bacteria, preparation of plasmid, EcoRI digestion, sequencing, and variation analysis were performed.
RESULTS AND CONCLUSION: The recombinant pGEM-T-CPT-ⅡE4 was proved to contain whole sequence of CPT-Ⅱ-E4 with 1305 nucleotides which could code 435 amino acids. According to the original sequence from Genebank, two variable sites were found at 1618 (GTC→ATC) and 1858 (TTT→TCT) which code amino acids of V368I and F448L respectively.

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