Preparation Of Monoclonal Antibody And Screening Of Phage Display Single-Chain Antibody To Zearalenone

Zearalenone (ZEN) is produced by several Fusarium species wich mainly contaminates grains including corn, barley, wheat, oats, sorghum. ZEN wich has significant estrogenic-like effect can cause reproduction toxicity, immunity toxicity, liver toxicity, genotoxic activity. It also has potential carcinogenicity. ZEN can threaten human health and the development of animal husbandry by contmainating cereals and their products, milk, meet and so on.Prevention and control the danger of ZEN in time is important by the effective detection technologies. In GB13078.2-2006, the content of ZEN in the formulated feed and corn should be controlled to500μg/kg. The new national standard GB2761-2011gives rule that the content of ZEN in cereals and their products should be controlled to60μg/kg.In oder to stop the threat of ZEN from the origins, we should detecte the content of ZEN accurately and effectively according to GB13078.2-2006and GB2761-2011. In recent years, more and more technologies are used to the detection of ZEN, such as high performance liquid chromatographic (HPLC), thin layer chromatography (TLC), high performance liquid chromatography-mass spectrometry, immunoassay. Immunoassay can be used to screen a large number of samples, meanwhile, it is widely applied now for its sensitive, special, rapid.The high quality antibody is necessary to immunoassay. In this study hybridomas producing anti-ZEN monoclonal antibody were obtained, the RNA of ZEN is extracted from the hybridomas, finally, by genetic engineering technology, positive clones wich express single-chain antibody to ZEN were obtained. The study lays the foundation for getting the specific antibody to ZEN immunoassay by genetic engineering antibody.Test I The preparation of the monoclonal antibodies against zearalenone. ZEN was dissolved in pyridine and reacted with carboxymethoxylamine to synthesize ZEN oximation (ZEN-CMO). The absorption was tested to caculate the synthesis rate of ZEN-CMO using ultraviolet spectrum scan method. The conjugation of bovine serum albumin (BSA) and CMO-ZEN was performed via activated esters. The conjugation of OVA and ZEN was performed via formaldehyde condensation. The complete antigen and coating antigen were identified by ultraviolet spectrum. Babl/c mouse was immunized first by intraperitoneal routes, and boosted by intrasplenic immunization. After the fusion of spleen and Sp2/0, positive cells were screened by indirect ELISA, indirect competitive inhibition enzyme immunoassay was used for the specific detection of positive cells. The results showed that ZEN-CMO was successfully synthesized, the ratio of ZEN-CMO reached65%. The complete antigen and coating antigen were successfully synthesized. One monoclonal cell line named4C8was successfully obtained. The group of anti-ZEN monoclonal antibody was IgG2b and the light chain was κ. Its chromosome average number was found to be104. Two peptide chains were presented of anti-Mab in SDS-PAGE, about25kDa and50kDa, respectively. The titer of culture medium supernate was1:512, the titer of ascites was1:9600. The concentration of asciles protein was1.639g/L. The cell supernant is specific to ZEN. The results indicated that4C8can be used to prepare the single-chain antibody to zearalenone.Test II Screening and expression of a single-chain antibody to zearalenone. A total RNA was extracted from hybridoma cell line. The first strand of cDNA wich was taken as template was amplified by RT-PCR. Then the variable region genes of heavy chain and light chain (VH and VL) were amplified respectively from the first strand of cDNA by PCR. VH, VL and DNA linker encoding (Gly4Ser)3were linked forming single chain fragment of variable region (scFv) by splicing overlap extension PCR (SOE-PCR). sfi I and Not I digested the scFv gene was ligated into sfi I and Not I digested pCANTAB5E. The recombinant phagemid vector was transformed into E.coli TGI, then was rescued by the helper phage M13K07, a primary phage display library was constructed. Using ZEN-OVA as coating antigen. After three rounds of enrichment screening, the antibody titer was up to3.7x106cfu/mL, the enrich factor exceeded66.2. Using phage-ELISA, three strains of antigen-positive clone were screened from enriched clone. Positive recombinant phages were used to infect the E.coli HB2151. The recombinant vector was transformed into the E.coli BL2151. After induced by IPTG, a soluble recombinant protien with the molecular of approximately35kDa was detected by SDS-PAGE. The expression level of the soluble protein was analyzed by using BandScan softerware. Acorrding to the expression level, the best induced condtions were determined, the best induced temperature was30℃, the best induced time was4h, the best IPTG induced concentration was0.8mmol/mL. Western Blot proved good specificity of the recombinant protien. It was concluded that the soluble expression of single chain antibody to ZEN was successful and specific.