| Mycotoxins are a range of toxic secondary metabolites produced by various funguses and the contamination of grains and all kinds of agricultural products, which exist in nature widely. Mycotoxins have been a potential threat to human health and the development of animal husbandry that may not only reduce the quality of the products, but also lead to serious foodborne poisoning and cause people or animals to acute or chronic poisoning. Animal tests showeded that the animals that took in mycotoxins contaminated feed would have some pathological changes, such as immune suppression, tissue necrosis, liver and kidney damage, reproductive disorder, carcinogenesis and cacogenics and so on, and make direct or indirect impact on the health of humans and animals. Many kinds of mycotoxin exist in nature with complex toxicities. They have caused highly concerns over global food security owing to the widely pollution and toxicity. In order to prevent mycotoxins contaminated food and animal products from the human food chain, directly or indirectly, strengthening the detection of mycotoxins is essential. In recent years, the detection technologies for mycotoxins have been developed rapidly, such as the chromatographic detection technology mainly based on high-performance liquid chromatography (HPLC) and immunology detection technology based on enzyme-linked immunosorbent assay (ELISA). Though high sensitivity, chromatography analytical method has shortcomings, such as the high quality requirement for sample and complex pretreatment operation, which make it difficult to be applied in actual production extensively. Compared to this, immunological method, based on the antigen-antibody has been favored by people due to its easy operation and fast advantages. Now, some experts and scholars focus on the various aspects of optimization and improvement of ELISA method in order to improve the detection performance.Genetically engineered antibody is the third generation of antibody that developed on the application of recombinant DNA technology and protein engineering technology and based on gene structure and function of the antibody. The significance of the genetically engineered antibody is to obtain the specific engineered protein with a series of operations, such as cutting, splicing and modification for antibody genes. Compared with polyclonal antibody and monoclonal antibody, genetically engineered antibody have its advantages, for example, short time prepare, simple operation, scale production and low cost. The Single-chain antibody (ScAb) is a member of genetic engineering antibody, which has been regarded as one direction of immunoassay technology development. As the improved product of traditional monoclonal antibody, ScAb has a better application prospect because of its stable and conserving easily. In this study, two kinds of ScAb against two common mycotoxins, ochratoxin A (OTA) and fumonisin B1(FB1) were obtained with molecular biology technique and phage display technique, so as to achieve a breakthrough both on theory and on technology, and would be the basic work for its immunoassay application and development.Test I Synthesis and characterization of complete antigen for fumonisin B1and ochratoxin AIn order to obtain FB1and OTA antibody genes, FB1was coupled with carrier proteins ovalbumin (OVA) and bovine serum albumin (BSA) respectively, by one step glutaraldehyde method, while two complete antigens FB1-OVA and FB1-BSA were synthesized. OTA was coupled with OVA and BSA respectively to synthesized complete antigens OTA-BSA and OTA-OVA by carbodiimide method. Ultraviolet absorption spectroscopy (UV), gel electrophoresis, fourier transforms infrared spectroscopy (IR) and matrix-assisted laser absorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to determine the coupled effects and coupling ratios of the complete antigens, respectively. FB1-BSA and OTA-BSA were used to immunize BALB/c mice, and FB1-OVA and OTA-OVA were used as solid phase coating antigens, respectively. The indirect enzyme-linked immunosorbent assay (i-ELISA) was used to determine the titer of anti-serum from mice. The results showed that carrier proteins coupled with FB1and OTA successful, respectively. The coupling ratios of FB1-BSA, FB1-OVA, OTA-BSA and OTA-OVA were10:1,5:1,3:1and9:1by MALDI-TOF-MS method, respectively. The titers of anti-serum form the mice immunized FB1-BSA or OTA-BSA could be reached1:1.024×105determined by i-ELISA method, respectively. The complete antigens and the mice immunized could be used for the next test. Test II The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against fumonisin B1A total RNA was extracted from hybridoma cell line F3that secreted FB1monoclonal antibody. The first strand of cDNA was amplified by reverse transcription PCR (RT-PCR). The heavy chain and light chain variable region genes(VH and VL) were amplified by PCR used cDNA as the template, respectively. The FB1single chain antibody variable region gene fragment (ScFv) was splicing by overlap extension PCR (SOE-PCR) using a flexible the polypeptide (Gly4Ser)3as DNA linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into Escherichia coli (E. coli) host strain TG1and then rescued by M13K07helper phage. Biopanning and screening, FB1-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of FB1ScAb with IPTG. The results showed that FB1murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were348and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was717bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TG1, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.1×1015cfu·mL-1. After five times biopanning enrichment and screening, five strains that could secrete the FB1-specific phage single-chain antibody were selected successfully. Transformed cells were then infected with M13K07helper phage to rescue the phagemid with its ScFv gene insert. Recombinant phages which were produced contain a single-stranded DNA copy of the phagemid and antibody ScFv gene and display one or more copies of the recombinant antibody at their tips. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.Test IH The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against ochratoxin AA total RNA was extracted from spleen cell of mice immunized by OTA-BSA. The first strand of cDNA was amplified by RT-PCR. The VH and VL were amplified by PCR used cDNA as the template, respectively. The OTA ScFv was splicing by SOE-PCR using a linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into E. coli TGI and then rescued by M13K07helper phage. Biopanning and screening, using OTA-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of OTA ScAb with IPTG. The results showed that OTA murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were369and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was738bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TGI, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.65×1016cfu·mL-1. After five times biopanning enrichment and screening, three strains that could secrete the OTA-specific phage single-chain antibody were selected successfully. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.Test IV Characterization of single-chain antibody against fumonisin B1and ochratoxin AThe immune activities of OTA and FB1recombinant ScAb were detected by Western blotting (WB) and i-ELISA, respectively. The titers of OTA and FB1recombinant ScAb were detected by i-ELISA, respectively. The sensitivities and competitive inhibition standard curves of OTA and FB1recombinant ScAb were established by competitive indirect enzyme-linked immunosorbent assay (ci-ELISA), respectively. And the specificities and cross-relativities of OTA and FB1ScAb to test toxins were determined using FB2, FB3, AFB1, ZEA and DON, respectively. The results showed that good immune activities of OTA or FB1ScAb to antigen were proofed through WB and i-ELISA method. The best working concentration of FB1-OVA antigen was5μg·mL-1and the best dilution of FB1ScAb was1:1600. The FB1ScAb titers reached104. The FB1competitive inhibition curve was established by ci-ELISA method, the linear equation y=-16.663x+117.98(R2=0.9674) and50%inhibition concentration (IC50) for OTA was4.08μg·mL-1, ranging from0.31～20μg·mL-1. The cross-reactivity inhibition rates of FB1ScAb to FB2and FB3were71.7%and87.6%, and the significant cross-reactivity with DON, AFB1, OTA and ZEA. The FB1ScAb has the better activity, sensitivity and specificity. The best working concentration of OTA-OVA antigen was10μg·mL-1and the best dilution of OTA ScAb was1:800. The OTA ScAb titers reached103. The OTA competitive inhibition curve was established by ci-ELISA method, the linear equation y=16.075x+116.84(R2=0.9797) and IC5o for OTA was4.16μg·mL-1, ranging from0.31～10μg·mL-1. The OTA ScAb had little cross-reactivity with DON, AFB1and ZEA. The OTA ScAb has better activity, sensitivity and specificity. |
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