Expression Of ScFv-AP Fusion Proteins Against Fusarium Mycotoxins

Fusarium head blight （FHB） or scab caused by species of Fusarium is an economically important disease on small grain cereal crops, prevalently in the areas with warm temperature and high humidity worldwide. In China, epidemics of FHB occur frequently in the middle and downstream regions of the Yangtze River and in the Heilongjiang province in the northeastern region. Modern methods like high performance liquid chromatography （HPLC）, gas chromatography （GC）, thin layer chromatography （TLC）, high performance liquid Chromatography-mass spectroscopy （HPLC -MS）, and immunobased assays such as ELISA are now largely used in detection of these mycotoxins.In this research, we design and synthesize oligonucleotide fragments with the selected E. coli-preferred codon according to the amino acid sequence of anti-ZEN scFv and anti-DON scFv, then synthesize the gene of Vh and VL by overlap extension PCR. The V_H-Linker-V_L, namely svFv was prepared ligasing the V_H and V_L genes with （Gly₄Ser）₂ Linker. The scFv-alkaline phosphatase fusion gene were subcloned into bacterial expression vector and yeast expression vector, respectively. The scFv-alkaline phosphatase were expressed in E. coli and yeast, and were assayed the activity of protein.The results indicated that, we obtained the right anti-ZEN scFv and anti-DON scFv genes by SOE-PCR, and constructed the scFv-AP fusion genes. The scFv-AP gene were expressed in E. coli. Analysis of SDS-PAGE analysis showed that recombinant fusion proteins were successfully expressed and most of them existed in the form of inclusion body, but we detected that some soluble protein in the supernatant by ELISA and purificated by affinity chromatography. Activity identification of scFv-AP showed that only anti-ZEN scFv-AP had AP enzyme activity. So scFv and alkaline phosphatase were linked via a linker sequence coding （Gly₄Ser）₃. The genes coding for the fusion protein was constructed into bacterial expression vector. The expression of the fusion protein was confirmed by ELISA detection, Activity Identification of scFv-AP showed that anti-DONGS-AP and D2GS -AP had AP enzyme activity. Detection of the cell wall protein of Fusarium graminearum with D2-AP demonstrated that the fusion protein had activity of both single chain antibody and alkaline phosphatase.We cloned the fusion genes into yeast expression vector pPIC9K. Then the recombined vectors were transformed into Pichia Pastoris GS115 by electroporation. To use G418 sereened positive clone, whether gene of fusion gene integrate into genome of Piehia pastoris with PCR identified. The positive clone expressed in Piehia Pastoris trains. Western blot showed that anti-ZEN-AP, D2-AP was expressed in theyeast Pichia pastoris sueeessfully. These establish the foundation of application of the fusion protein.