Screening And Expression Of A Single-chain Fv Antibody Against Clenbuterol

At present, the issues of food safety are due to biological hazards, chemical hazards and other safety problems. Chemical hazards are the most prominent, and run throughout the industrial chain of agriculture including agricultural production, processing and storage. The development of modern society has brought rich, high-yield agricultural products. However, unreasonable use of pesticides, fertilizer, veterinary drugs during planting and aquaculture has also led to excessive residues and harmed human health. One of the key technologies for the prevention is to establish a rapid detection method. At present, the used rapid detection methods include enzymology, immunology and so on. The key issue for establishing rapid detection methods is to get antibodies.Phage Display Antibody Library is a new method for generation of monoclonal antibodies that mimics the natural selective system of immunity with the development of genetic engineering technology. Particular advantages of phage display methods, compared with hybridoma technology, is that the phage antibody library can mimic natural antibody process, and human and animals does not need to be immunized, and that it can change antibody affinity by simulating the antibody affinity mature process, PCR mispairing and site-directed random mutation. Therefore the phage display antibody library technology has the huge potential to enhances the antibody affinity.Although new technologies to enhance the antibody affinity emerge unceasingly, however, how to obtain the high affinity antibody is still one of essential problems of antibody library research determining if genetic engineering antibody can be used.Recently the research of small molecule chemical antibody has been reported frequently, but the methods using the phage antibody library technology have rarely been reported. The construction of pesticide, and veterinary medicine antibody preparation technology platformis the foundational and key problem of the establishment of the standard antibody libraries and the rapid detection methods. Because small molecule pesticideand veterinary medicine do not have immunogenicity, the conventional method is very difficult to obtain the ideal antibodies. Therefore establishing one kind of optimized and new technical systems is the important guarantee to obtain the high quality monoclonal antibodies . In order to establish the methods of small molecular weak antigens antibody preparation , This research aims to screen single chain Fv antibodies against small molecule chemicals by phage display antibody library technology using CLB as the model. We report as follows:The first part: screening phage single chain Fv antibody against Clenbuterol antigenClenbuterol is one ofβ2 - acceptor agonist . The chemical structure is stable, so to destroy half of CLB structure in polluted meat production,such as pig liver must fry in oil for 5min at 126℃t. CLB was prohibited to be used in our country from 1997. Therefore, for establishing CLB rapid detection method, we mainly use the phage surface demonstration technology to obtain the CLB specific antibody. After 3 times of enriched procedure in the order of adsorption-elution-amplification, we obtained specific anti-CLB single chain Fv antibody The main results were as follows:1. 5 CLB-positive phage scFv clones were screened after 3 times of enriched procedure in the order of adsorption-elution-amplification from semi-synthesized phage single chain Fv libraries.2. The ELISA and the competitive inhibition ELISA results proved it has specificity.3. The fragment size after NotⅠ/SfiⅠenzyme digestion is consistent with the anticipated size.The second part: Screening anti-CLB phage single chain antibodies using biotinylation CLB as target antigen.A streptavidin has 4 binding sites for the biotin, and its binding constant is 1015 mol/L which is approximately 10,000 times more than the binding constant between antigen and antibody. The research of this part is to screen scFv antibodies from the phage antibody library using using biotinylation CLB as target antigen.In order to avoid non-specificity antibodies, we designed two schemes: first, it was to screen antibodies directly from the phage antibody libarary; second, to avoid non-specific antibodies, we let biotin and streptavidin act with the phage antibody libarary. Then we screened the antibodies useing solid phase incubation and liquid phase incubation The main results were as follows:1. 5 phage antibody clones were obtained, the ELISA results proved that it had the good affinity and the competitive inhibition ELISA results proved it had strong specificity. 2. 4 phage antibody clones were obtained using solid phase binding, and 5 phage antibody clones were obtained using liquid phase binding. The ELISA and the competitive inhibitio ELISA results proved it had very good specificity.3. After NotⅠ/SfiⅠenzyme digestion, the fragment size obtained is consistent with the anticipated fragment size.The third part: expression of anti-CLB single chain Fv antibody in E.coliAt present, there are many kinds of antibody expressing systems such as bacterium, yeasts and mammal cells which have their respective advantages and disadvantages. For single chain Fv antibodies, E. coli expressing system is the best. After analysis of the results we have obtain, it is obvious that the single chain Fv antibodies screened using biotinylation CLB have strong specificity. Therefore the expression of a scFv antibody clone which has the strongst specificity was carried out. The main results were as follows:1. Expression of pHEN1-CLB1 was selected.2. Recombinant plasmid was constructed after the scFv gene fragment was subcloned into expression plasmid , and was renamed as 5E-BHNS-CLB1. Then the plasmid was transformed into BL-21 E.coli.3. After identification of 5E-BHNS-CLB1 by PCR amplification and enzyme digestion, the fragment size was consistent with the anticipated size, and was registered EU681272 on GenBank.4. SDS-PAGE and Western blot results showed that its molecular weight size is consistent with the anticipated size, and it can bind with the anti-E-tag label antibody. These data suggested that the screened scFv antibody had been expressed.The fourth part: screening scFv antibodies against other chemicals The scFv antibodies against other chemicals such as arilin, permethrin and melamine were screened through the phage display technology. The results were as follows:1. 16 positive phage scFv antibody clones against the three molecules were screened after 3 times of enriched procedure in the order of adsorption-elution-amplification from semi-synthesized phage single chain Fv libraries using the method in the first part. The ELISA and the competitive inhibitio ELISA results proved they had very good specificity. 2. 5 phage antibody clones were obtained against biotinylated melamine after 3 times of enriched procedure using the method in the second part. The ELISA and the competitive inhibitio ELISA results proved they had very good specificity.