硫代磷酸酯类杀虫剂ScFv抗体库的构建与初步筛选

Construction and Preliminary Screening of a Single Chain Variable Fragment Library against Thio-phosphate Pesticides

旨在构建硫代磷酸酯类杀虫剂单链抗体(ScFv)库,并对其进行初步筛选。采用RT-PCR法从牛血清蛋白(BSA)人工抗原免疫的Balb/C小鼠脾细胞中克隆出VH和VL基因,通过重叠延伸PCR方法将VH和VL基因拼接在一起,构建ScFv基因,与表达载体pCANTAB 5E连接后,在大肠杆菌中诱导表达。采用亲和富集法,经6轮淘筛后用酶联免疫吸附分析(ELISA)测定其活性。得到滴度为2.2×106 pfu/mL噬菌体库,ELISA检测呈阳性。说明成功表达并筛选得到鼠源抗硫代磷酸酯类杀虫剂ScFv抗体库。

To construct and preliminarily screen a singe chain variable fragment antibody （ScFv） library against thio-phosphate pesticides. VH and Vc genes were cloned from the spleen cells of the Balb/C mouse, which was immunized by generic hapten of thio-phosphate pesticides conjugated to bovine serum albumin （BSA）. ScFv gene was spliced by sequence overlap extending PCR, and then it was ligated into pCANTAB 5E vector. The recombinant plasmid was transformed into E. coli and ScFv was induced by IPTG. After six rounds of panning, the bio-activity was tested by enzyme-linked immunosorbent assay （ELISA）. A ScFv antibody library with the titer of 2.22 X 10^6 pfu/mL was obtained, which showed positive identified by ELISA analysis. In conclusion, the mouse special ScFv antibody library against thio-phosphate pesticide was constructed successfully.