生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 17-.doi: 10.3969/j.issn. 2095-1736.2021.05.017

• 研究报告 • 上一篇    下一篇

三疣梭子蟹IAG基因的原核表达及质谱鉴定

  

  1. 宁波大学 海洋学院，宁波 315211
  • 出版日期:2021-10-18 发布日期:2021-10-20
  • 通讯作者: 朱冬发，博士，教授，研究方向为发育生物学和遗传育种学，E-mail：zhudongfa@nbu.edu.cn
  • 作者简介:脱　萍，硕士，研究方向为水生生物发育学，E-mail：757193591@qq.com
  • 基金资助:
    国家自然科学基金项目（41776165，31802265）

Prokaryotic expression and mass spectrometry determination of IAG gene in Portunus trituberculatus

  1. School of Marine Sciences, Ningbo University, Ningbo 315211, China
  • Online:2021-10-18 Published:2021-10-20

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摘要： 基于已知的三疣梭子蟹（Portunus trituberculatus）胰岛素样促雄腺激素（IAG）序列，对其ORF区域进行扩增，并连接至pET-22b、pET-28a和pET-28a-sumo等不同的原核表达载体中，随后分别转化至大肠杆菌BL21（DE3）与Rosetta（DE3）。通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达带有His标签的IAG重组融合蛋白，发现仅pET-28a-sumo-IAG有可见表达，且转化至Rosetta（DE3）菌株的IAG重组蛋白表达量较高。进一步对Rosetta-pET-28a-sumo-IAG的表达条件进行优化，发现IPTG终浓度为0.6 mmol/L，16 ℃诱导16 h后重组蛋白表达量最大。制备的融合蛋白主要以可溶性形式高效表达，通过HisTrap HP 柱进行纯化后，经Western Blot分析发现该融合蛋白可与鼠抗His-tag多克隆抗体发生特异性结合。将制备的蛋白进行质谱分析，得到的2条肽段能够分别与三疣梭子蟹的B链和A链相匹配，表明重组融合蛋白表达成功。

关键词: 三疣梭子蟹, 胰岛素样促雄腺激素基因, 原核表达, 质谱鉴定

Abstract: Based on the known insulin-like androgenic gland hormone (IAG) gene in Portunus trituberculatus nucleotide sequence, the ORF region of Pt-IAG was amplified, liganded into different prokaryotic vectors, including pET-22b, pET-28a and pET-28a-sumo, and then transformed into Escherichia coli BL21 (DE3) and Rosetta (DE3) strains, respectively. The His-tag labeled IAG recombinant fusion protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the SDS-PAGE found that only the pET-28a-sumo-IAG could be detected, and the IAG transformed into the Rosetta (DE3) strain was highly expressed. Further exploration of the condition of this Rosetta-pET-28a-sumo-IAG system showed that the IAG recombinant fusion protein was most efficiently expressed when induced by 0.6 mmol/L of IPTG at 16 ℃ for 16 h. The fusion protein was mainly expressed in soluble form and was further purified by HisTrap HP column. Western Blot analysis showed that the fusion protein could be recognized by mouse anti-His-tag. The purified protein was further analyzed by mass spectrometry, and two peptides could be matched to the B chain and A chain, respectively, which indicated that the recombinant fusion protein was successfully obtained.

Key words: Portunus trituberculatus, insulin-like androgenic gland hormone gene, prokaryotic expression, mass spectrometry determination

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