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Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性

徐舒怡, 李雅贤, 胡海, 张莉, 边延林, 朱建伟, 吴明媛

徐舒怡, 李雅贤, 胡海, 张莉, 边延林, 朱建伟, 吴明媛. Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性[J]. 中国药科大学学报, 2021, 52(5): 622-629. DOI: 10.11665/j.issn.1000-5048.20210516
引用本文: 徐舒怡, 李雅贤, 胡海, 张莉, 边延林, 朱建伟, 吴明媛. Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性[J]. 中国药科大学学报, 2021, 52(5): 622-629. DOI: 10.11665/j.issn.1000-5048.20210516
XU Shuyi, LI Yaxian, HU Hai, ZHANG Li, BIAN Yanlin, ZHU Jianwei, WU Mingyuan. Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody[J]. Journal of China Pharmaceutical University, 2021, 52(5): 622-629. DOI: 10.11665/j.issn.1000-5048.20210516
Citation: XU Shuyi, LI Yaxian, HU Hai, ZHANG Li, BIAN Yanlin, ZHU Jianwei, WU Mingyuan. Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody[J]. Journal of China Pharmaceutical University, 2021, 52(5): 622-629. DOI: 10.11665/j.issn.1000-5048.20210516

Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性

基金项目: 国家自然科学基金资助项目(No.81773621);转化医学国家重大科技基础设施(上海)开放课题基金资助项目(No.TMSK-2020-131)

Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody

Funds: This study was supported by the National Nature Science Foundation of China (No.81773621) and the National Facility for Translational Medicine (Shanghai) Open Research Program (No.TMSK-2020-131)
  • 摘要: 针对细胞程序性死亡-配体1(PD-L1)和CXC趋化因子受体4型(CXCR4)两个靶点,设计anti-PD-L1&CXCR4双特异性纳米抗体的基因序列,C末端连接组氨酸标签(6 × His标签),通过pET-22b(+)重组表达质粒转化大肠埃希菌E.coli BL21,经 IPTG 诱导表达,以可溶性形式存在于菌体裂解上清液。为了提高双特异性纳米抗体的产量和纯度,采用3种不同的方法进行样品制备和纯化。结果表明,通过机械裂菌并改进缓冲液的盐离子和咪唑浓度以及pH,经His Trap FF亲和色谱柱纯化后,对双特异性纳米抗体分离效果较好,目的蛋白产量超过1 mg/L,纯度可达到97%。同时,anti-PD-L1&CXCR4双特异性纳米抗体能够与细胞表面两个抗原特异性结合,增强IL-2活化的人外周血单个核细胞(PBMC)对胰腺癌细胞株 AsPC-1的杀伤能力,为其后续体内药效学评价奠定了基础。
    Abstract: Targeted programmed death-ligand 1 (PD-L1) and CXC chemokine receptor type 4 (CXCR4), gene sequences encoding anti-PD-L1 nanobody and anti-CXCR4 nanobody were cloned into the pET-22b (+) vector to construct recombinant expression plasmid of anti-PD-L1&CXCR4 bispecific nanobody, which was connected with 6 × His tag and transformed into E.coli BL21 (DE3). The expressed proteins were then found to exist as a soluble form in the supernatant of bacterial lysate after induction of IPTG.Three purification methods were used to obtain the target protein in order to improve the yield and purity of the bispecific nanobody.The bacterial supernatant was separated and purified by His Trap FF affinity chromatographic column.The target protein output could exceed 1 mg/L, and the product purity could reach up to 97%.Besides, the anti-PD-L1&CXCR4 bispecific nanobody shows a specific binding ability to two antigens on the cell surface, enhancing the cytotoxicity of IL-2 activated human peripheral blood mononuclear cells (PBMC) to tumor cell line AsPC-1, which lays the foundation for further evaluation of its drug efficacy in vivo.
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出版历程
  • 收稿日期:  2021-03-01
  • 修回日期:  2021-09-25
  • 刊出日期:  2021-10-24

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