| Fusarium head blight of wheat is one of the most severely diseases in China. Above all the methods to control the disease, the benzimidazole fungicides (eg carbendazim) were commonly employed as the most effective measures.However, Fusarium head blight pathogen population of resistance to benzimidazole fungicides develops rapidly during recent years basing the monitoring research. In our lab, we have found that the resistance of pathogen to carbendazim (MBC) is mainly caused by β2-tubulin gene point mutations in Fusarium graminearum. Mutation at codon167,198,200and other amino acid would result in resistance, respectively.In this paper, expression vectors of mutant β2tubulin gene at different codons were constructed by the method of prokaryotic expression. And monoclonal antibody of β2tubulin in wild-type strain was eatablished. So the above materials were prepared for investigate the principle of resistance and also the interaction between protein and MBC.First, in sensitive strains of Fusarium graminearum, β1tubulin sensitive gene (β1-S) and β2tubulin sensitive gene (P2-S) were amplified, respectively. In moderate resistance strains and higher resistance strains, β2tubulin moderate resistance gene (β2-MR) and P2tubulin higher resistance gene (β2-HR) were amplified, respectively. After sequenced, prokaryotic expression vectors β1-S/pET28a, β2-S/pET28a, β2-MR/pET28a, β2-HR/pET28a were constructed and then transformed into Escherichia coil Rosetta (DE3)pLysS, respectively. The products were all fused6×His purification tag in C-terminal after inducted by IPTG (1mM). With the help of analysis by SDS-PAGE electrophoresis and Western blot, it was showed that the fusion proteins, with a relative molecular weight of50KD, were expected to accurately express in E.coli, and specific reaction with anti-His tag monoclonal antibody.However, in the system of prokaryotic expression, the target protein was so much that would present in the inclusion bodies as the expression vector could be at high level expression. On the other hand, the absence of modification during prokaryotic expression would aslo result in inclusion bodies, As it is known to us, for lack of biological activities, inclusion bodies should be dissolved, renaturation and purification before the further reasearch. In the text, we chose β2-S tubulin as the material, and optimized the washing conditions of inclusion bodies. And aslo two protein renaturation methods (dialysis refolding and diluted-dialysis refolding) and three protein purification strategies (His-Trap Hp pre-column purification, ion exchange purification and PD-10gravity-column purification) were compared, respectively. At last the washing buffer at3M urea was used to wash the inclusion bodies; however, protein refolding should be after diluted-dialysis. And PD-10gravity-column purification was the first choise of purification.After refolding and purification, β2-S was used to immunize mouse. About4weeks later, when the mouse anti-serum titer was1:6500, the spleen cells of immunized mouse were fused with SP2/0murine myeloma cells by PEG. ELISA assay was performed several times with purified β2-S when hybridoma cells occurred. Hybridoma cells, H1, H2, H3was subcloned, and four monoclonal antibodies (Mab1-1, Mab1-2, Mab2-1, Mab3-1) were got. The refolding-and purified-β1-S, β2-MR and β2-HR were prepared individually just as the β2-S. The four different fusion proteins were used to detect the specificity of monoclonal antibodies by Western blot and ELISA. It was showed that four different monoclonal antibodies could bind to β2-S and β2-HR but β1-S and β2-MR. The results may indicate that β2-S, β1-S and β2-MR could be distinguished by the four different monoclonal antibodies. |
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