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  • ISSN 1006-3080
  • CN 31-1691/TQ

溶组织梭菌胶原酶H在大肠杆菌中的分泌表达

赵钱山 刘晓 李素霞

赵钱山, 刘晓, 李素霞. 溶组织梭菌胶原酶H在大肠杆菌中的分泌表达[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.2021092400
引用本文: 赵钱山, 刘晓, 李素霞. 溶组织梭菌胶原酶H在大肠杆菌中的分泌表达[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.2021092400
ZHAO Qianshan, LIU Xiao, LI Suxia. Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.2021092400
Citation: ZHAO Qianshan, LIU Xiao, LI Suxia. Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.2021092400

溶组织梭菌胶原酶H在大肠杆菌中的分泌表达

doi: 10.14135/j.cnki.1006-3080.2021092400
详细信息
    作者简介:

    赵钱山(1995—),男,安徽合肥人,硕士生,研究方向重组蛋白酶。E-mail:3170430889@qq.com

    通讯作者:

    李素霞,E-mail: lisuxia@ecust.edu.cn

  • 中图分类号: Q78

Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli

  • 摘要: 溶组织梭菌Clostridium histolyticum胶原酶H(ColH)作用于胶原蛋白的Y-Gly处,将其水解成小分子肽。通过在colH基因中融合大肠杆菌外膜蛋白A的信号肽序列,成功构建了分泌高分子量的ColH(116 kDa)的菌株,结果发现信号肽在N端的位置影响其分泌效果,N端若有多余的氨基酸片段会显著降低信号肽引导胶原酶的分泌功能。通过正交实验与单因素实验对诱导条件和培养基添加剂优化提高其分泌表达量, 结果为:在诱导温度为25 ℃、诱导菌浓OD600=0.9、IPTG(异丙基- β -D-硫代吡喃半乳糖苷)浓度为0.1 mmol/L、装液量为20%,镁离子浓度为10 mmol/L,诱导2.5 h后添加2%甘氨酸条件下,诱导培养20 h,胞外胶原酶活性最高为0.68 U/mL,是优化前的38.1倍,显著提高了分泌表达量。研究结果还表明,镁离子的添加可增加ColH的胞外分泌。

     

  • 图  1  构建的两种ColH分泌质粒结构示意图

    Figure  1.  Schematic representation of two ColH secretory expression plasmids

    图  2  3种重组菌株胶原酶的胞外分泌情况

    Figure  2.  Extracellular secretion of collagenase in the three recombinant strains

    图  3  正交实验的因素指标趋势图

    Figure  3.  Factor index trend chart of orthogonal experiment

    图  4  SDS-PAGE电泳分析胶原酶ColH的分泌表达鉴定

    Figure  4.  SDS-PAGE analysis of culture medium components and intracellular soluble components

    图  5  甘氨酸添加量对ColH表达和分泌的影响

    Figure  5.  Effects of amount of glycine added on ColH expression and secretion

    图  6  甘氨酸添加时间对ColH表达和分泌的影响

    Figure  6.  Effect of time of glycine added on ColH expression and secretion

    图  7  钙离子对ColH表达和分泌的影响

    Figure  7.  Effects of calcium ion on ColH expression and secretion.

    图  8  镁离子对ColH表达和分泌的影响

    Figure  8.  Effects of magnesium ion on ColH expression and secretion.

    图  9  诱导时间对ColH表达和分泌的影响

    Figure  9.  Effect of inducing time on ColH expression and secretion.

    表  1  本研究使用的引物

    Table  1.   Primers used in this work

    Primer namesPrimer sequence(5′–3′)
    F1ATCGAGGATCCATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGG
    TTTCGCTACCGTAGCGCAGGCCGTCCAGAACGAAAGCAAACGCTAC
    R1ATCGACTCGAGACGACCAACGCTGCCTTC
    F2AAGGAGATATACCATGAAAAAGACAGCTATCGCGATTGC
    R2GGTGGTGGTGCTCGAGACGAC
    Note: The underlined part is the restriction site, the oblique part is the signal peptide sequence, and the bold part is the homologous sequence of primer and carrier terminal
    下载: 导出CSV

    表  2  因素水平表

    Table  2.   Factor level table

    FactorsLevels
    Inducing
    temperature/℃
    c(IPTG)/
    (mmol·L−1)
    OD600Loading
    volume fraction/%
    1200.10.4510
    2250.50.9020
    3301.01.8030
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-09-24
  • 网络出版日期:  2022-04-12

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