抗黄曲霉毒素B1纳米抗体的原核表达、纯化及活性分析

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、pH7.4、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/mL,线性范围为0.95 ～ 42.45 ng/mL.%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 pET25b(+) to generate the recombinant expression vector pET25b(+)-G8,which was transformed into E.coli BL21 (Rosetta,DE3) for expression.The effects of temperature,methanol concentration,ionic strength and pH 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 devel oped using the purified recombinant G8 for detecting of AFB1.Under optimized conditions,the half inhibitory concentration (IC50) of the ELISA was 4.61ng/mL,and the liner range was 0.95 ～ 42.45 ng/mL.