杀虫剂甲萘威纳米抗体的制备及鉴定

Preparation and identification of a nanobody for the insecticide carbaryl

为制备杀虫剂甲萘威特异性的纳米抗体,首先合成甲萘威完全抗原XH_3-KLH,免疫羊驼,然后提取外周血淋巴细胞中总RNA,PCR扩增获得羊驼重链抗体的可变区基因片段VHH,与噬菌粒载体pComb3X连接后电转化到E.coli ER2738感受态细胞中,构建了库容量为6.7×10~7cfu的纳米抗体文库。以XH_3-BSA复合物为模板,经过四轮筛选,从构建的噬菌体展示纳米抗体库中淘选得到1种特异性结合甲萘威的纳米抗体C3-1,在大肠杆菌Top10F’中表达并纯化,经间接竞争的酶联免疫吸附分析法(enzyme-linked immuno sorbent assay,ELISA)测定甲萘威的半数抑制质量浓度IC_(50)为179.7μg/L。该纳米抗体为后续甲萘威的免疫分析方法建立以及环境样品检测提供了基础。

Insecticide carbaryl as the goal to prepare nano specific antibody,firstly,the carbaryl complete antigen XH_3-KLH was synthesized,an alpaca was immunized,and then the total RNA was extracted from alpaca's peripheral blood lymphocytes and VHH genes were PCR amplified.The VHH fragment was cloned into a plasmid pComb3 Xand then electrotransformed to E.coli ER2738.The capacity of the constructed library was 6.7×10~7cfu.After four rounds of biopanning,one clone C3-1highly selective to carbaryl was isolated from the constructed library using conjugate XH_3-BSA as the coating antigen.This VHH(C3-1),also known as nanobody,was expressed under the induction of IPTG and employed to develop an ELISA for carbaryl.The half maximum inhibition concentration(IC_(50))of carbaryl was 179.7ng/mL.The nanobody proved to be a potential for the detection of carbaryl in the environment sample.