黄鳍鲷白细胞介素1β基因2种表达载体的构建

李建柱, 江世贵

李建柱, 江世贵. 黄鳍鲷白细胞介素1β基因2种表达载体的构建[J]. 南方水产科学, 2005, 1(3): 37-41.
引用本文: 李建柱, 江世贵. 黄鳍鲷白细胞介素1β基因2种表达载体的构建[J]. 南方水产科学, 2005, 1(3): 37-41.
LI Jian-zhu, JIANG Shi-gui. The construction of two recombinant expression vectors of interleukin-1β gene from Acanthopagrus latus[J]. South China Fisheries Science, 2005, 1(3): 37-41.
Citation: LI Jian-zhu, JIANG Shi-gui. The construction of two recombinant expression vectors of interleukin-1β gene from Acanthopagrus latus[J]. South China Fisheries Science, 2005, 1(3): 37-41.

黄鳍鲷白细胞介素1β基因2种表达载体的构建

基金项目: 

广东省“十五”攻关项目 99B06103G

详细信息
    作者简介:

    李建柱(1976-),男,硕士研究生,研究方向为海洋生物技术。E-mail: li_jianzhu@163.com

    通讯作者:

    江世贵,E-mail: jiangsg@21cn.com

  • 中图分类号: Q782

The construction of two recombinant expression vectors of interleukin-1β gene from Acanthopagrus latus

  • 摘要:

    根据黄鳍鲷白细胞介素1β(interleukin-1β, IL-1β)基因全长cDNA序列(GenBank登录号为AY669059)设计、合成1对特异性引物,扩增编码黄鳍鲷IL-1β基因前体肽的基因序列,通过T-A克隆构建了克隆载体pMD18T-IL1β。以克隆载体pMD18T-IL1β为模板,以设计合成的带酶切位点的引物分别扩增黄鳍鲷IL-1β的前体肽和预测的成熟肽基因序列,经BamHI和SalI双酶切后将其插入表达载体pQE30中,构建了原核表达质粒pQE30-pIL1β和pQE30-mIL1β。经酶切、PCR鉴定并最终通过序列测定表明,基因已正确插入到载体的多克隆位点,序列和读码框都正确无误,为黄鳍鲷IL-1β基因的体外重组表达研究打下了基础。

    Abstract:

    A pair of primers were designed and synthesized according to the interleukin-1β (IL-1β) gene sequence (GenBank accession number is AY669059) of the yellowfin seabream Acanthopgrus latus (Houttuyn). The plasmid pMD18T-IL1β was constructed after amplifying open reading frame cDNA sequence of the IL-1β gene by RT-PCR. The sequence of pro-peptide and predicted mature peptide were linked into expression vector pQE30 and two recombinant plasmids pQE30-pIL1β and pQE30-mIL1β were constructed. Restriction analysis, PCR amplification and sequencing demonstrated that the target fragments were inserted into pQE30 correctly.

  • 图  1   表达载体pQE30-pIL1β和pQE30-mIL1β的构建示意图

    Figure  1.   Construction of recombinant expression plasmid pQE30-pIL1β and pQE30-mIL1β

    图  2   重组质粒pMD18T-IL1β的酶切鉴定

    M.100 bp DNA分子量标准; 1.BamHI+SalI消化后的重组质粒

    Figure  2.   Identification of recombinant plasmid pMD18T-IL1β

    M.100 bp DNA marker; 1.Digested recombinant plasmid pMD18T-IL1β by BamHI and SalI

    图  3   重组质粒pQE30-pIL1β (a)和pQE30-mIL1β (b)的酶切鉴定

    M.100bp DNA分子量标准, 1. BamHI+SalI消化后的重组质粒;2. BamHI+SalI消化后的载体pQE30

    Figure  3.   Identification of recombinant plasmid pQE30-pIL1β and pQE30-mIL1β

    M. 100 bp DNA marker; 1.Digested recombinant plasmids by BamHI SalI; 2.Digested pQE30 by BamHI and SalI

    图  4   黄鳍鲷IL-1β氨基酸序列与其它鱼类IL-1β氨基酸序列CLUSTAL比对分析结果

    箭头所指为推测的成熟蛋白切点,相同和相似氨基酸残基分别用“*”和“.”或“: ”表示

    Figure  4.   Alignment of the predicated yellowfin seabream IL-1β translation with other known IL-1βs

    Identical (*)and similar (: or.)residues identified by CLUSTAL are indicated, predicated mature protein cut site is indicated with an arrow.

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出版历程
  • 收稿日期:  2005-05-08
  • 刊出日期:  2005-07-19

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