基于纳米抗体-荧光素酶的黄曲霉毒素B1检测方法构建  

Development of the Detection Method for Aflatoxin B1 Based on Nanobody-NanoLuciferase Fusion Proteins

作　　者：陈文星 王凤华 谭晓亮 晏石娟 张军[1] 吴绍文 Chen Wenxing;Wang Fenghua;Tan Xiaoliang;Yan Shijuan;Zhang Jun;Wu Shaowen(College of Resources and Environmental Sciences,Gansu Agricultural University,Lanzhou 730070;State Key Laboratory of Swine and Poultry Breeding Industry,Guangdong Provincial Key Laboratory for Crop Germplasm Resources Preservation and Utilization,Agro-biological Gene Research Center,Guangdong Academy of Agricultural Sciences,Guangzhou 510640)

机构地区：[1]甘肃农业大学资源与环境学院,兰州730070 [2]猪禽种业全国重点实验室广东省农作物种质资源保存和利用重点实验室广东省农业科学院农业生物基因研究中心,广州510640

出　　处：《中国食品学报》2024年第4期349-360,共12页Journal of Chinese Institute Of Food Science and Technology

基　　金：广州市基础研究计划基础与应用基础项目(SL2022A04J00899);国家自然科学基金项目(22307022);甘肃省自然科学基金项目(23JR RA1413);甘肃省科技厅重点研发计划-农业领域(23YFNA0036)。

摘　　要：黄曲霉毒素B1(AFB1)是一种剧毒、致癌的食源性污染物,严重威胁食品安全和公共健康。为开发快速检测AFB1的生物发光酶联免疫分析(BLEIA)方法,系统测试了3种抗AFB1特异性纳米抗体(NB)与纳米荧光素酶(Nluc)融合蛋白(G8-Nluc、Nluc-NB26和Nluc-NB28)的可溶性表达、纯化情况及酶催化活性。基于3种融合蛋白分别建立BLEIA检测体系,选择Nluc-NB26用于谷物样品的分析和验证。结果表明,Nluc-NB26的可溶性表达量最高,稳定性更好,Nluc-NB28的可溶性表达量次之,而G8-Nluc基本不可溶。不同表面活性剂对G8-Nluc的促溶解性研究表明,添加N-月桂酰肌氨酸钠可显著提高其溶解度,纯化得到的3种融合蛋白均具有良好的酶活性及抗原结合活性。基于融合蛋白的BLEIA检测结果显示,Nluc-NB28-BLEIA、G8-Nluc-BLEIA和Nluc-NB26-BLEIA体系检测AFB1的IC_(50)值分别为4.213,1.697,2.169 ng/mL,表明G8-Nluc-BLEIA体系的灵敏度最高,Nluc-NB26-BLEIA与前者接近,Nluc-NB28-BLEIA最低。综合考虑融合蛋白的可溶性表达量、稳定性及检测性能,对Nluc-NB26-BLEIA开展实际样品的分析和验证,结果表明:这一方法检测谷物中AFB1的平均回收率在91.1%~104.1%之间,与商业化酶联免疫吸附测定试剂盒结果相似,而检测的时间和试剂成本明显降低。研究结果为开发快速、高灵敏的AFB1检测技术提供参考。Aflatoxin B1(AFB1)is a highly toxic and carcinogenic foodborne contaminant that seriously threatens food safety and public health.To develop a rapid bioluminescent enzyme immunoassay(BLEIA)for detecting AFB1,this study systematically evaluated the soluble expression,purification,and enzymatic activity of three anti-AFB1 nanobody-nano luciferase fusion proteins(G8-Nluc,Nluc-NB26,and Nluc-NB28).Based on the three fusion proteins,BLEIA assays were established,and Nluc-NB26 was selected for the analysis and validation of cereal samples.The results indicated that Nluc-NB26 exhibited the highest soluble expression level and better stability,followed by Nluc-NB28,while G8-Nluc was largely insoluble.Testing with various surfactants revealed that adding sodium lauroyl sarcosinate improved the solubility of G8-Nluc significantly.The purified fusion proteins all exhibited suitable enzymatic and antigen-binding activities.BLEIA results based on the fusion proteins showed the IC_(50) values for detecting AFB1 were 4.213,1.697 and 2.169 ng/mL for the Nluc-NB28-BLEIA,G8-Nluc-BLEIA and Nluc-NB26-BLEIA systems,indicating that G8-Nluc-BLEIA had the highest sensitivity,comparable to Nluc-NB26-BLEIA,while Nluc-NB28-BLEIA had the lowest.Considering the soluble expression level,stability,and detection performance of the fusion proteins,Nluc-NB26-BLEIA was further applied to analyze and validate cereal samples.The results demonstrated that this method achieved average recovery rates of 91.1%to 104.1%,comparable to commercial ELISA kits,but with significantly reduced detection time and reagent cost.These findings offer valuable insights for developing rapid and highly sensitive detection techniques for AFB1.

关 键 词：黄曲霉毒素B1 纳米抗体 纳米荧光素酶 快速检测 生物发光酶联免疫分析 

分 类 号：TS207.5[轻工技术与工程—食品科学] O657.3[轻工技术与工程—食品科学与工程] TS201.6[理学—分析化学]