拟除虫菊酯类农药单链可变区抗体的制备、 鉴定及其特异性分析

Preparation, characterization and specificity analysis of single-chain fragment variable antibody against pyrethroid pesticides

中文关键词:  拟除虫菊酯类农药  单链可变区抗体  多残留免疫分析

英文关键词:pyrethroid pesticides  single-chain variable fragment  multi-residual immunoassay

中文摘要:

      目的 制备拟除虫菊酯类农药可溶性单链可变区(single-chain fragment variable, scFv)抗体, 鉴定其免疫活性并对其特异性进行分析。方法 构建表达载体pET28a(+)-scFv和pET42a(+)-scFv并转化BL21(DE3), 重组菌经诱导后通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定目的蛋白的表达。可溶性融合蛋白scFv/GST经Glutathione Sepharose 4B纯化后用Factor Xa切除GST标签获得scFv, 然后采用Western blotting 法对其进行鉴定。scFv对7种拟除虫菊酯类农药的特异性采用间接竞争酶联免疫法进行分析。结果 经0.1 mmol/L IPTG诱导后, 重组菌BL21(DE3)-pET28a(+)-scFv和BL21(DE3)-pET42a(+)-scFv表达的目标蛋白分别为包涵体抗体和部分可溶性抗体。经鉴定, 可溶性scFv相对分子量约为30 kDa, 效价为1:25600。同源半抗原Hap1对单链抗体scFv的IC50为2.43 μg/mL, 检出限为0.045 μg/mL; scFv抗体对氯菊酯、氯氰菊酯、溴氰菊酯均有较高特异性, IC50分别为3.29、4.72和5.55 μg/mL。结论 本研究成功获得了拟除虫菊酯类农药的可溶性单链可变区抗体, 该抗体对3种拟除虫菊酯农药具有很强的特异性, 该研究可为拟除虫菊酯类农药多残留免疫分析提供依据。

英文摘要:

      Objective To prepare the soluble single-chain fragment variable (scFv) antibody against pyrethroid pesticides, identify its immune activity and analyze its specificity. Methods The recombinant expression vectors pET28a(+)-scFv and pET42a(+)-scFv were transformed into BL21(DE3). The target proteins were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after induction of recombinant bacteria. scFv/GST was purified by Glutathione Sepharose 4B, and scFv was obtained after excision of GST with Factor Xa, and then identified by Western blotting. The specificity of scFv against seven pyrethroid pesticides was analyzed by indirect competitive-enzyme linked immunosorbent assay (IC-ELISA). Results After the recombinant strains BL21 (DE3)-pET28a(+)-scFv and BL21 (DE3)-pET42a(+)-scFv were induced by 0.1 mmol/L IPTG, the target proteins expressed in the form of inclusion body and partially in the soluble form, respectively. After being cleaved the GST-Tag, the soluble fusion protein gave out 30 kDa-scFv, and its titer reached 1:25600. The IC50 of the homologous hapten1 to scFv was 2.43 μg/mL and the detection limit was 0.045 μg/mL. Specificity analysis showed that the IC50 of scFv to permethrin, cypermethrin and deltamethrin were 3.29, 4.72 and 5.55 μg/mL, respectively. Conclusion In this study, a soluble scFv antibody against pyrethroid pesticides was successfully obtained with high specificity to three pyrethroids, which can provide the basis for multi-residual immunoassay of pyrethroid pesticides.