| Fumonisins are a group of mycotoxins maily produced by Fusarium verticillioides and Fusarium proliferatum. Fumonisins usually contaminates maize, sorghum, rice and other crops and products. Fumonisins have potential carcinogenicity and also can cause human and livestock poisoning, fumonisin B1 as the most toxic in fumonisins had been classified as class 2B Carcinogen by the International Agency for Research on Cancer(IARC). Thus, the guidance levels for fumonisins in human foods and animal feed have been proposed by FDA and European Comminity. Therefore, the development of rapid, accurate detection technolog of fumonisin and analysis of the main types of fusarium toxins in China, has important significance for ensuring food safety and health of human and livestock.Aflatoxins produced by Aspergillus flavus and Aspergillus parasiticus have a stong carcinogenic and teratogenic effects, had been classified as class I carcinogen by the International Agency for Research on Cancer(IARC). Aspergillus flavus is the predominant producer of aflatoxins. Specific antibodies of Aspergillus flavus can be used for immune detection of toxinic strains, and also can be used for the prevention or elimination of toxinic strains and aflatoxins. Therefore, expression and analysis of specific antibodis based on Aspergillus flavus strains screened in China, will provide information for further utilization of these antibodies.The experiment focuses on fumonisin immune and chemical detection and Aspergillus flavus antibody expression research, the main research results are as follows:1. Identify the type of strains by fusarium-specific primers, including F.temperatum, F.subglutinans and F.verticillioides. The genes related to fumonisins synthesis are detected by PCR, and the results show that the gene only exist in the F.verticillioides. The strains belong to F.temperatum, F.subglutinans do not contain the gene.2. We inoculated three different Fusarium spp. in maize medium to detect the types and concentration of mycotoxins. The toxin produced by three Fusariums in maize culture are detected using liquid chromatography tandem mass spectrometry(LC-MS/MS) and enzyme-linked immunoabsorbent assay(ELISA). We found that the reusts from the two different detection methods are consistent, which also is corresponding with the results of the molecule detection. Above all, between the three Fusarium, only F.verticillioides produce fumonisins.3. High-affinity single-chain antibody 3(sc Fv3) and sc Fv12 were fused to generate dimeric-antibody with three kinds of short connecting peptides(218linker,G4 S,(G4S)3). The fused dimeric-antibody was overexpressed in bacteria and then purified using affinity chromatography. SDS-PAGE and western blot analysis showed that three dimericantibodies were successfully expressed, these antibodies can lay the foundation of aflatoxin detection and analysis.4. Antibacterial peptides(MSI99 and AFP2) and single-chain antibodies(sc Fv3 and sc Fv12) were used to construct plant expresstion vectors in different combinations. Those vectors had been transformed into Arabidopsis by Agrobacterium mediated method. We successfully obtain transgenic Arabidopsis,and after cultivation in greenhouse and PCR identification of each generation, transgenic plants have been training to the third generation.5. Semi-quantitative PCR was used to demonstrate the gene expression in transcription level in the T3 transgenic Arabidopsis. The results showed that the genes of antibacterial peptides, single-chain antibodies and fution antibodies were transcripted. In addition, we explored the Arabidopsis thaliana leaves inoculating method with Aspergillus flavus spores in vitro, and we found that Aspergillus flavus spores can infect Arabidopsis leaves without extension. |
PDF Full Text Request