基于纳米抗体-碱性磷酸酶融合蛋白的一步酶联免疫吸附分析法检测黄曲霉毒素B_1  

One-step Enzyme Linked Immunosorbent Assay for Detection of Aflatoxin B_1 Using a Nanobody-Alkaline Phosphatase Fusion Protein

作　　者：曹冬梅[1] 许杨[1,2] 涂追[1] 李燕萍[2] 熊亮[1,3] 付金衡[2] 

机构地区：[1]南昌大学食品科学与技术国家重点实验室,南昌330047 [2]南昌大学中德联合研究院,南昌330047 [3]赣南医学院预防医学系,赣州341000

出　　处：《分析化学》2016年第7期1085-1091,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.31301479);南昌大学食品科学与技术国家重点实验室青年研究基金(No.SKLF-QN-201513);江西省自然科学基金重大项目(No.20152ACB20005)资助~~

摘　　要：从噬菌体展示的抗黄曲霉毒素B1(AFB_1)纳米抗体文库中,通过四轮亲和淘选,得到抗AFB_1纳米抗体G8。将编码纳米抗体G8的基因与碱性磷酸酶(AP)基因融合,构建了重组融合表达载体pET25b(+)-G8-AP,将其转化大肠杆菌BL21(Rosetta,DE3)感受态细胞,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导融合基因表达。SDS-PAGE结果表明,融合蛋白G8-AP为可溶性表达,Ni^(2+)-NTA亲和层析柱纯化融合蛋白,对硝基苯磷酸二钠(pNPP)法测得纯化后的G8-AP的碱性磷酸酶比活力为(364.5±8.3)U/mg。ELISA检测结果表明,G8-AP具有特异识别AFB1的活性,与其它真菌毒素(AFB_2、AFG_1、AFG_2、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮、伏马菌素B_1)无交叉反应。基于G8-AP建立了检测AFB1的一步ELISA法。在优化的条件下,即甲醇浓度20%、盐离子浓度20 mmol/L、pH 7.4条件下,方法的半数抑制浓度(IC_(50))为19.8 ng/mL,线性范围为4.3~92 ng/mL,检出限为2.6 ng/mL。对玉米和小麦样品加标回收实验结果表明,基质对本方法的干扰不明显,可用于实际样品检测。A phage clone （designated as the anti-aflatoxin B1 nanobody, G8） specific binding to aflatoxin B1 （AFB1 ） was screened from an immunized nanobody phage library. The DNA fragment encoding G8 was subcloned into the vector pET25b（＋）-alkaline phosphatase （AP）, and fused to the N terminal of AP. The fusion protein G8-AP was expressed in E. coli BL21 （Rosetta, DE3） as soluble protein, and purified by immobilized metal affinity chromatography. The purified G8-AP exhibited AP activity of 364.5 ±8.3 U/mg. Enzyme-linked immunosorbent assay （ELISA） showed that G8-AP was also capable of recognizing AFBl , with negligible cross-reactivity toward other six common mycotoxins. A one-step ELISA was established to detect AFB1 using G8-AP. The effects of methanol concentration, ionic strength, and pH on the detection sensitivity of the G8-AP-ELISA were evaluated and optimized. Under the optimized conditions （methanol 20%, NaCI 20 mmol/L, pH 7.4）, the half inhibitory concentration （ IC50 ） of the G8-AP based ELISA was 19.8 ng/mL, with the linear range of 4.3-92 ng/mL and the detection limit of 2.6 ng/mL. The recoveries of AFB1 for corn and wheat samples were 90.4% - 101.5%.

关 键 词：抗黄曲霉毒素B_1纳米抗体 碱性磷酸酶 融合表达 酶联免疫吸附分析 

分 类 号：TS207.3[轻工技术与工程—食品科学]