Selection Of Antibodies Against Pyrethroids And Antibodies Against E. Coli And Their Activity Analysis

Pyrethroids and E. coli were selected for studying the pesticide residue and pathogenic microorganism in the food safety problems, we screened antibodies of them, and studied the biological activities of the antibodies. Because pyrethroids are well known for lower toxicity and easily decomposing in the past few years, they were widely used in the agricultural pesticides control in the worldwide. At present, researches indicate that long-lasting exposure to pyrethroids will be a threat to human health. Prevention and control of harmful microorganism pollution in food is another research hotspot in the food safety field, and E. Coli is the most typical. Phage display technology which have been developed in recent years provides a powerful tool and method for high throughput screening, a variety of antibodies of antibiotics, pesticides and toxins et al. have been obtained by phage display technology. Rarely research on the screening the broad spectrum antibody of pyrethriod and antibacterial proteins from the naive human phage display library has been reported.In this study, PBA, broad spectrum monoclonal antibody of pyrethroids and E.coli were used as antigen respectively, the naive human phage display antibody library was used to screen antibodies of them. Then, the biological activities of obtained antibodies were studied. The main studies are as follows:1. Screening, identification and activity analysis of the domain antibody of pyrethroidsFive domain antibodies, which were capable of binding to the phenoxybenzoic acid(PBA), were obtained after four rounds of screening domain antibody (DAB) library using PBA as the antigen. Competitive ELISAs were constructed based on the five domain antibodies. ELISA based on A3 has the highest sensitivity with strong identification capability to cypermethrin, beta-cypermethrin, and fenvalerate, weak identification capability to flucythrinate, and no identification capability to fenpropathrin, deltamethrin and permethrin. The IC50 were 2.586 ?g/mL, 1.814 pg/mL and 2.251 ?g/mL to cypermethrin, beta-cypermethrin and fenvalerate, respectively. Meanwhile, the developed ELISA process was successfully applied to fortified Chinese cabbage samples, the results showed that the recoveries of cypermethrin, beta-cypermethrin and fenvalerate ranged from 89.4% to 91.5%. After verified by molecular docking and alanine-scanning mutagenesis,the results showed that the residues of Ile 55, Val 74, Pro 76 and Lys 125 involved in the combination of the PBA and A3.2. Screening, identification and activity analysis of the anti-idiotype antibodyA naive human phage display library was used to screen anti-idiotype antibody of pyrethroids, three domain antibodies (A8, B8, and C6) which were able to identify monoclonal antibody were obtained. And the competitive ELISAs were constructed,respectively. ELISA based on A8 has the highest sensitivity with strong identification capability to Cypermethrin, Beta-Cypermethrin and Fenvalerate, weak identification capability to flucythrinate, and no identification capability to fenpropathrin, deltamethrin and permethrin. The IC50 were 1.775 pg/mL,1.624 ?g/mL and 3.675 ?g/mL to Cypermethrin, Beta-Cypermethrin and Fenvalerate, respectively. Meanwhile, the developed ELISA process was successfully applied to fortified Chinese cabbage samples, and the results showed that the recoveries of cypermethrin,beta-cypermethrin,and fenvalerate ranged from 87.4% to 90.2%. The antigen structure of A8 was predicted.3. Screening, identification and soluble expression of ScFv against E. coliTomlinson I library was used to screen the ScFv against E. coli for four rounds with whole cell and outer membrane protein alternately as screening antigen. A1, A3, B4, D5,which bound to E. coli were obtained and successfully expressed in BL21-pET26b system.Al and B4 exhibited the antibacterial activity against E. coli by agar well diffusion assays.Optimization expression conditions of A1 were induction temperature 25 ?, IPTG concentration 1.0 mmol/L, induction time 15 h. Optimization expression conditions of B4 were induction temperature 30?, IPTG concentration is 0.6 mmol/L, induction time 21 h.His-trap FF purification column was used to purificate the ScFv.4. Preliminary study of inhibitory activity and mechanisms of ScFv B4The MICs of the ScFv B4 against Escherichia coli, Yersinia enterocolitica, Salmonella typhimurium, Bacillus cereus were 128 ?g/mL, 256 ?g/mL, 128 pg/mL,256 ?g/mL respectively. ScFv B4 didn't show the antibacterial activity against Staphylococcus aureus,Saccharomyces cerevisiae. By studying the antibacterial activity, stability and hemolytic activity of ScFv B4 on E. coli, the results showed that with the concentration of ScFv B4 increases, the growth of E. coli was distinctly inhibited. The ScFv B4 showed low stability to heat, and the antibacterial activity was no significant difference under the neutral condition, but antibacterial activity was obviously decreased in acid or base condition. The ionic strength decreased antibacterial activity; the papain, trypsin, alkaline protease and proteinase K. could destroy ScFv B4; in addition ScFv B4 did not show hemolytic activity.The antibacterial mechanism of action of ScFv B4 was studied on E. coli. Effect of morphology of the E. coli treated by ScFv B4 was studied by SEM analysis, the results showed that the morphology of the E. coli was collapsed and distorted, the longer process time the more seriously. We studied the metabolic activity of E. coli, using the methods of MTT to determine the generation of formazan, and we found that the ScFv B4 affected the metabolic activity of E. coli. Rhodamine 123 was used as fluorescent indicator to study the the member potential, the results indicated that the the member potential of E. coli was decreased after ScFv B4 treatment. By measuring OD260nm, we analyzed the exosmosis of macromolecular substances which has ultraviolet absorption, we can learn that with the increase of ScFv B4 concentration and treatment time the exosmosis was more obvious.After analyzing the results of the flow cytometry, we found that the cell membrane of E.coli which treated by ScFv B4 was broken down.