抗赭曲霉毒素A五价纳米抗体的表达及分析  

Expression and Analysis of Pentavalent Nanobody against Ochratoxin A

作　　者：张俊毅 陈敏达 涂追[2] 许杨[2,3] 邓建中 李燕萍[2,3] ZHANG Jun-yi1 , CHEN Min-da1 ,TU Zhui2, XU Yang2,3 ,DENG Jian-zhong1 ,LI Yan-ping2,3(1.Institute of Food Science and Technology, Nanchang University, Nanchang 330047, China ; 2.State Key Laboratory of Food Science and Technology, Nanehang University, Nanchang 330047, China; 3.Sino- Germ any Joint Research Institute, Nanchang University, Nanehang 330047, China)

机构地区：[1]南昌大学食品学院,江西南昌330047 [2]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [3]南昌大学中德联合研究院,江西南昌330047

出　　处：《食品工业科技》2018年第21期224-229,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金(31471648);江西省科研攻关计划(20171BBG70072)

摘　　要：利用霍乱毒素B亚基(B subunit of Cholera toxin,CTB)能自组装形成五元环状结构,将霍乱毒素B亚基与纳米抗体(VHH28)进行融合从而达到纳米抗体多聚化的目的。将重组载体p ET25b(+)-CTB-VHH28转化至大肠杆菌BL21(DE3)中,IPTG诱导表达,对诱导剂浓度、诱导温度、诱导时间进行优化,SDS-PAGE凝胶电泳分析融合蛋白表达情况。结果显示CTB-VHH28为可溶表达,其中单体分子量大小约30 kDa,五聚体分子量大小约150 kDa,均与预测的分子量大小一致;确定了其最优表达条件为:IPTG浓度为0.05 mmol/L,诱导温度为16℃,诱导时间为10 h。经过镍柱纯化后成功得到了纯度较高的五价纳米抗体,表达量为20 mg/L。对其活性进行测定,结果表明CTB-VHH28能与赭曲霉毒素A(ochratoxin A,OTA)特异性结合,在2.5%甲醇条件下,间接竞争ELISA半抑制浓度(IC_(50))为0.38 ng/mL。热稳定性及甲醇耐受性结果显示,CTB-VHH28在25~55℃具有良好的热稳定性,反应体系中甲醇浓度低于20%时,竞争抑制率变化不大,具有较好的甲醇耐受性。结论:本研究所制备的五价纳米抗体可以用于OTA检测。B subunit of Cholera toxin canself-assemble to form a five- membered structure and be fused with nanobody toaehieve the purpose of muhi-nanobody.The constructed expression vector pET25b（ ＋ ）-CTB-VHH28 was transformed into Escherichia.coli BL21 （DE3）and induced with IPTG for expression. The IPTG concentration, expression temperature and expression time were optimized and the obtained recombinant proteins were subjected to SDS-PAGE. SDS-PAGE assay was used to analyze recombinant protein expression, and the molecular weight of CTB-VHH28 monomer is 30 kDa and that of pentavalentis 150 kDa, which are consistent with predicted molecular weight. By optimizing the concentration of IPTG, the expression temperature and the expression time, the optimal expression conditions was： the concentration of IPTG was 0.05 mmol/L, the induction temperature was 16 ℃ and the induction time was 10 h.After purification by nickel column, high- purity pentavalent nanobody was successfully obtained and the production yield was 20 mg/L.CTB-VHH28 could specifically bind to OTA by activity measurement and direct ELISA assay.By the activity assay,results showed that the fusion proteins CTB -VHH28 could specifically bind to OTA and the IC_50 of indirect competitive inhibition ELISA was 0.38 ng/mL under the condition of 2.5% methanol.Besides, CTB-VHH28 performed excellent thermal stability in a temperature of 25～55 ℃, and showed little change in competition inhibition rate when methanol concentration was no more than 20% in the reaction system demonstrating a good tolerance toward methanol.In summary ,the pentavalent nanobody prepared in this study can be used for OTA detection.

关 键 词：赭曲霉毒素A 纳米抗体 霍乱毒素B亚基 原核表达 

分 类 号：Q51[生物学—生物化学]