生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (5): 15-.doi: 10.3969/j.issn.2095-1736.2020.05.015

• 研究报告 • 上一篇    下一篇

肝靶向肽修饰的人内皮抑制素rES-CSP融合蛋白可溶性表达及活性鉴定

  

  1. 广东药科大学 生命科学与生物制药学院 广东省生物活性药物研究重点实验室， 广州 510006
  • 出版日期:2020-10-18 发布日期:2020-10-14
  • 通讯作者: 马艳，高级实验师，硕士生导师，研究方向为药用生物活性物质的开发与应用，E-mail:mayan820901@126.com
  • 作者简介:余田甜，硕士研究生，研究方向为药用生物活性物质的开发与应用，E-mail:1946597106@qq.com
  • 基金资助:
    国家自然科学基金项目(No.81502520)；广东省自然科学基金项目(No.2016A030310299)；广东省科技计划项目（2017A09090544）

Soluble expression and biological activity identification of CSP I-plusmodified human endostatin rES-CSP

  1. Guangdong Key Laboratory of Pharmaceutical Bioactive Substance, College of Life Science and  Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China
  • Online:2020-10-18 Published:2020-10-14

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摘要： 为了方便获得大量的有活性rES-CSP融合蛋白，通过优化诱导表达条件，实现融合蛋白rES-CSP的可溶性表达，并纯化后检测其生物学活性，使该融合蛋白作为药用蛋白大规模生产成为可能。首先在常规条件下诱导表达融合蛋白rES-CSP，通过SDS-PAGE电泳分析，选出最优菌株；继而优化诱导温度和诱导时间，Western Blot检测目的蛋白可溶性表达情况，Ni-NTA亲和层析柱进行纯化后RP-HPLC分析其纯度； CCK-8细胞增殖实验和流式细胞仪检测融合蛋白生物学活性。结果选出目的蛋白约占菌体总蛋白43.84%的单克隆菌株为研究对象，在诱导温度为25 ℃，诱导时间为20 h时，融合蛋白rES-CSP实现可溶性表达，并且可溶性表达量较高。经纯化后获得的rES-CSP融合蛋白纯度达到98%以上；制备的rES-CSP融合蛋白具有抑制肝癌细胞HepG2增殖作用，且与HepG2结合能力较强；为融合蛋白rES-CSP的应用研究奠定基础。

关键词: 可溶性表达, 肝靶向肽修饰的人内皮抑制rES-CSP, 活性鉴定

Abstract: The soluble expression of the fusion protein rES-CSP was accomplished by condition optimization in order to obtain amount of active protein conveniently. Its biological activity was detected after purification. The fusion protein was made possible as a pharmaceutical. The rES-CSP was expressed under normal conditions, and the optimal strain was selected by SDS-PAGE. After optimization of induction time and temperature, the targeted protein was analyzed by Western Blot, purified by Ni-NTA, and the purity of the fusion protein was analyzed by RP-HPLC. The biological activity of fusion protein was detected by CCK-8 and flow cytometry. Results showed that the monoclonal strain with the target protein of about 43.84% of the total bacterial protein was selected as the research object. Soluble fusion protein rES-CSP was expressed at 25 ℃ for 20 h, and the amount was higher. The purity of rES-CSP after purification was over 98%. Biological assay showed that rES-CSP inhibited the proliferation of hepatoma cell line HepG2 and had stronger binding ability to HepG2.

Key words: soluble expression, CSP I-plus modified human endostatin rES-CSP, biological activity identification

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