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中国精品科技期刊2020
吴箫,梁赟,钟于红,等. 基于SpyCatcher/SpyTag的ZEN/DON抗独特型纳米抗体的体外自组装[J]. 华体会体育,2024,45(24):153−162. doi: 10.13386/j.issn1002-0306.2024020014.
引用本文: 吴箫,梁赟,钟于红,等. 基于SpyCatcher/SpyTag的ZEN/DON抗独特型纳米抗体的体外自组装[J]. 华体会体育,2024,45(24):153−162. doi: 10.13386/j.issn1002-0306.2024020014.
WU Xiao, LIANG Yun, ZHONG Yuhong, et al. In Vitro Self-assembly Study of ZEN/DON Anti-idiotype Nanobody Based on SpyCatcher/SpyTag[J]. Science and Technology of Food Industry, 2024, 45(24): 153−162. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2024020014.
Citation: WU Xiao, LIANG Yun, ZHONG Yuhong, et al. In Vitro Self-assembly Study of ZEN/DON Anti-idiotype Nanobody Based on SpyCatcher/SpyTag[J]. Science and Technology of Food Industry, 2024, 45(24): 153−162. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2024020014.

基于SpyCatcher/SpyTag的ZEN/DON抗独特型纳米抗体的体外自组装

In Vitro Self-assembly Study of ZEN/DON Anti-idiotype Nanobody Based on SpyCatcher/SpyTag

  • 摘要: 为了提升真菌毒素抗独特型纳米抗体的免疫分析性能,并实现双特异性检测抗原的模拟,本研究以玉米赤霉烯酮(Zearalenone,ZEN)、脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)的抗独特型纳米抗体N28、Z6为研究对象,将SpyCatcher/SpyTag标签与N28、Z6分别进行融合表达,在此基础上基于体外自组装的方式,开展N28、Z6的双价、双特异性抗独特型纳米抗体的构建研究。结果表明,实现了四种SpyCatcher/SpyTag标签融合抗独特型纳米抗体N28-SpyCatcher、N28-SpyTag、Z6-SpyCatcher、Z6-SpyTag的原核可溶表达。基于Z6-SpyCatcher、Z6-SpyTag所建立的竞争抑制标准曲线的IC50分别为0.18、0.20 ng/mL,其灵敏度高于单体Z6的IC50(0.28 ng/mL);SpyCatcher标签与N28融合后有利于N28检测灵敏度的提升,IC50值由94.39降低至42.33 ng/mL,而SpyTag标签与N28融合后,则抑制了其与DON抗体的结合。此外,在4 ℃反应12 h的简单反应条件下,基于SpyCatcher/SpyTag的体外自组装特性,成功制备了Z6、N28的双价、双特异性抗独特型纳米抗体Z6-Z6、N28-28与N28-Z6、Z6-N28。ELISA的鉴定结果表明,双价抗独特型纳米抗体均保留了与对应抗体的结合特性,其中N28-Z6还实现了同时与ZEN、DON抗体的结合活性,由于SpyTag标签与N28结合会阻碍其与DON抗体的结合,因此Z6-N28仅保留了与ZEN抗体结合的活性。综上,本研究基于SpyCatcher/SpyTag实现了ZEN/DON双价、双特异性抗独特型纳米抗体的简便快速制备,同时提示SpyCatcher标签对于ZEN/DON抗独特型纳米抗体的免疫分析性能提升具有促进作用。

     

    Abstract: To improve the immunoassay performance of mycotoxin anti-idiotype nanobody and to realize mimicry of bispecific detected antigens, in this study, anti-idiotype nanobody against zearalenone (ZEN) and deoxynivalenol (DON) were used, and the SpyCatcher/SpyTag tags were fusion-expressed with N28 and Z6, respectively, and based on the in vitro self-assembly, the construction study of bivalent and bispecific anti-idiotype nanobody of N28 and Z6 were carried out. The results showed that the prokaryotic soluble expression of four SpyCatcher/SpyTag labeled fusion anti-idiotype nanobody, N28-SpyCatcher, N28-SpyTag, Z6-SpyCatcher, and Z6-SpyTag were achieved. The IC50 of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL, respectively, and their sensitivities were significantly higher than that of the IC50 of monomeric Z6 (0.28 ng/mL). The fusion of the SpyCatcher tag with N28 was favorable for the enhancement of the sensitivity of the N28 assay, with the IC50 value decreased from 94.39 to 42.33 ng/mL, whereas the fusion of SpyTag label with N28 inhibited its binding to DON monoclonal antibody. In addition, bivalent and bispecific anti-idiotype nanobody Z6-Z6, N28-28, and N28-Z6, Z6-N28 of Z6 and N28 were successfully prepared based on the in vitro self-assembly property of SpyCatcher/SpyTag under the simple reaction condition of 4 ℃ for 12 h. The characterization results of the ELISA showed that the bivalent anti-idiotype nanobody retained their binding properties to the corresponding antibodies. The binding properties of the corresponding antibodies, in which N28-Z6 also achieved the binding activity with ZEN and DON antibodies simultaneously, and Z6-N28 only retained the binding activity with ZEN antibody because the binding of SpyTag tag to N28 would hinder its binding to DON antibody. In conclusion, the present study realized a simple and rapid preparation of ZEN/DON bivalent and bispecific anti-idiotype nanobody based on SpyCatcher/SpyTag and suggested that the SpyCatcher tag had a facilitating effect on the immunoassay performance enhancement of ZEN/DON anti-idiotype nanobody.

     

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