抗黄曲霉毒素B_1纳米抗体的原核表达、纯化及活性分析  

Prokaryotic expression,purification and bioactivity analysis of nanobody against AFB_1

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

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

出　　处：《食品与发酵工业》2016年第5期19-24,共6页Food and Fermentation Industries

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)

摘　　要：表达和纯化抗黄曲霉毒素B1（Aflatoxin B1,AFB1）纳米抗体（G8）,并分析纳米抗体的热稳定性及生物学活性,建立基于纳米抗体检测AFB1的ELISA方法。将编码抗AFB1纳米抗体的基因亚克隆至原核表达载体,转化至大肠杆菌BL21（Rosetta,DE3）,IPTG诱导表达后,SDS-PAGE分析蛋白表达情况;采用酶联免疫吸附法（enzyme-linked immunosorbent assay,ELISA）分析纳米抗体的热稳定性、有机溶剂耐受性、盐离子耐受性及耐酸碱性。结果显示,在大肠杆菌中成功表达可溶性的抗AFB1纳米抗体,表达量为80 mg/L,在25~65℃之间,抗体具有良好的热稳定性。在5%甲醇、p H7.4、10 mmol/L PBS条件下,建立了G8-ELISA检测AFB1的方法,该方法的半抑制浓度（IC50）为4.61 ng/m L,线性范围为0.95~42.45 ng/m L。To develop a nanobody-based ELISA for detection of AFB1,the anti-AFB1 nanobody G8 was expressed in Escherichia coli and bioactivity of purified recombinant protein was evaluated using indirect enzyme linked immunosorbent assay（ ELISA）. The DNA fragment encoding G8 was subcloned into the vector p ET25b（ ＋） to generate the recombinant expression vector p ET25b（ ＋）-G8,which was transformed into E. coli BL21（ Rosetta,DE3） for expression. The effects of temperature,methanol concentration,ionic strength and p H on the bioactivity of G8 were analyzed by ELISA. The results showed that nanobody against AFB1 was expressed in E. coli in soluble fraction with the production yield of 80 mg / L,and G8 was stable at temperature below 65 ℃. A nanobody based ELISA was developed using the purified recombinant G8 for detecting of AFB1. Under optimized conditions,the half inhibitory concentration（ IC50） of the ELISA was 4. 61 ng /m L,and the liner range was 0. 95 ~ 42. 45 ng /m L.

关 键 词：黄曲霉毒素B1 纳米抗体 原核表达 酶联免疫吸附法 

分 类 号：TS207.3[轻工技术与工程—食品科学]