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

作　　者：吴箫 梁赟 钟于红 郭原臻 黄斌辉 何庆华[2,3] WU Xiao;LIANG Yun;ZHONG Yuhong;GUO Yuanzhen;HUANG Binhui;HE Qinghua(College of Food Science&Technology,Nanchang University,Nanchang 330000,China;State Key Laboratory of Food Science and Resources,Nanchang University,Nanchang 330000,China;Sino German Joint Research Institute,Nanchang University,Nanchang 330000,China)

机构地区：[1]南昌大学食品学院,江西南昌330000 [2]南昌大学食品科学与资源挖掘全国重点实验室,江西南昌330000 [3]南昌大学中德联合研究院,江西南昌330000

出　　处：《食品工业科技》2024年第24期147-156,共10页Science and Technology of Food Industry

基　　金：江西省“双千计划”人才项目(JXSQ2023201029);江西省自然科学基金重点项目(20232ACB205027)。

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IC_(50)分别为0.18、0.20 ng/mL,其灵敏度高于单体Z6的IC_(50)(0.28 ng/mL);SpyCatcher标签与N28融合后有利于N28检测灵敏度的提升,IC_(50)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。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.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 IC_(50) of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL,respectively.Their sensitivities were significantly higher than that of the IC_(50) 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 IC_(50) 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 fa

关 键 词：抗独特型纳米抗体 SpyCatcher/SpyTag 真菌毒素 玉米赤霉烯酮 脱氧雪腐镰刀菌烯醇 

分 类 号：TS201.6[轻工技术与工程—食品科学]