| Pyrethroid insecticides are a broad spectrum of insecticides synthesized by natural pyrethrin as a precursor.Because of its light stability,high efficiency,low toxicity,long efficacy and other advantages,it is widely used in agriculture and forestry pests,household health and livestock and poultry breeding pest control and other fields.At present,pyrethroid insecticides have been used with organic phosphorus,carbamate pesticides and known as the most widely used three pesticides.Pyrethroids are a kind of neurotoxic agent,which are highly toxic to fish,bees and silkworms and they also are potential threat to the environment and human health.Studies have shown that pyrethroids are enriched in the human body by the food chain,which can cause acute poisoning or accumulation of toxicity,while the body's immune and cardiovascular system has a significant toxic effect.In recent years,the pyrethroid pesticide residues in agricultural products has gradually attracted people's attention,so the study of new residue detection technology for pyrethroid pesticide is of great significance.In this study,the soluble single chain variable region antibody against pyrethroid pesticide was expressed by genetic engineering technology.The method of enzyme-linked immunosorbent assay for pyrethroid insecticides was established,optimized and applicated initially.The main results are as follows:(1)The soluble single chain variable region antibody against pyrethroid pesticides has successfully obtained.The prokaryotic expression vectors pET28a(+)-scFv and pET42a(+)-scFv were successfully constructed and transformed into E.coli BL21(DE3).The results showed that the single chain antibody obtained by BL21(DE3)/pET28a(+)-scFv was inactive in inclusion body form.Recombinant strain BL21(DE3)/pET42a(+)-scFv was induced by 0.1 mM IPTG to obtain the soluble protein GST/ scFv at 15? for 15 h.The fusion protein GST/scFv was purified by glutathione sepharose GSTrap FF column,and then the GST tag was removed by factor Xa at 20? for 6h.The secondary purified product was analysised by SDS-PAGE and Western blotting.We found that the single chain variable region antibody in single band is about 28 KDa.The titer of the single chain variable region antibody was 1: 2560 identificated by indirect noncompetitive ELISA.(2)IC-ELISA method for permethrin,cypermethrin and deltamethrin was established.ODmax and IC50 used as the main parameters,the factors influencing the enzyme-linked immunoreactivity were optimized.The optimized result were as follow Hap1-BSA at 5?g/mL to coat microplate at 37?/2 h,single chain variable region antibody diluted by 640 times,the dilution is PBST(pH7.4)buffer with 10% methanol,the competition time is 80 min,HRP/Anti-His enzyme secondary antibody diluted by 7 000 times.The inhibition curves of hapten1 to single chain antibody were determined by the optimized conditions: I=24.463lgC+42.501,R2=0.9844,IC50 is 2.03?g/mL,the lowest detection limit(IC10)is 0.046?g/mL,linear detection range(IC20-IC80)is 0.12~34.11?g/mL.The results of cross-reaction showed that single chain antibody had strong recognitive effect to permethrin,cypermethrin and deltamethrin,and the IC50 are 3.29,4.73,5.55?g/mL respectively.The results also showed that buprenorphine,fenvalerate,Fenaprofen are recognizied weakly and permethrin is hardly recognizied.The recovery results in water showed that the recoveries of permethrin,cypermethrin and deltamethrin were between 82.60~104.01% when add 0.5~10?g/mL,and RSD were between 2.71~6.38%,indicating that the single chain antibody can be used to detecte those three kinds of pyrethroids in water samples.(3)The feasibility of IC-ELISA to determinate permethrin in Chinese cabbage was detected by GC.The results showed that the IC-ELISA recovery was 74.68%~82.97% and the RSD was 4.13%~11.34% at the add concentration of 0.156~5mg/kg.The correlation between the two methods was good,but we still need to explore how to eliminate the interference of Chinese cabbage substrate during the analysis process. |
PDF Full Text Request