The Heterologous Expreession Of Porcine Epidemic Diarrhea Virus COE Protein In Aspergillus Niger And Fusarium Venena Tum

Aspergillus niger is an industrial strain commonly used for the expression of heterologous proteins. Fusarium venenatum ATCC20334 is a new discovery of filamentous fungi having a high expression ability of protein. Porcine epidemic diarrhea virus (PEDV) is a swine influenza virus which can cause acute infection in piglets, the coe gene encode glycoprotein which can induce neutralizing antibodies in the hosts. Vaccine expression system of coe gene was constructed in Aspergillus niger ATCC1015 and Fusarium venenatum ATCC20334. The mainresults are as follows:(1) Respectively comparison the secreted protein background and the secreted protease activity of F.venenatum with A.niger and A.oryzae. The experimental results suggested that compared to A.niger and A.oryzae, F.venenatum secrete a low protein background. Activity of secreted protease in F.venentum culture broth was significantly lower than that in A.niger and A.oryzae culture broth.(2) Microscope was utilized to observe the fermentation morphology different between A.niger and F.venenatum in YPD and Vogel’s medium. The observation result showed that mycelium of A.niger in culture medium was compact pellets, F.venenatum was showed dispersed mycelium which is more conducive to the secretion of extracellular proteins. The fermentation morphology of A.niger or F.venenatum in YPD compared to Vogel’s medium was the same.(3) Applying genetic engineering techniques, and using the strong promoter gpdA, glaA and trypsin in the fungi hosts, we constructed vaccine expression vectors of coe gene on the plasmid pFGL59 containing Hyg resistant gene, and obtained A.niger and F.venenatum engineering strains with expression vectors by agrobacterium-mediated transformation. Extracellular proteins of engineering strains were detected by SDS-PAGE test, the results indicate that Coe protein was efficiently expressed, processed and secreted in F.venenatum host strains under the control of promoter trypsin. Coe protein was not detected in extracellular proteins of the strains containing gpdA and glaA promoters.(4) The experiment results showed that due to optimizing coe codons, under the control of promoter trypsin the production of Coe protein was significantly increased in F.venenatum and Coe protein was also expressed in A.niger and F.venenatum under the control of promoter glaA.In this research, A.niger and F.venenatum were utilized to construct the vaccine expression system of PEDV, successful expression of Coe protein validated the efficiency of this system. By optimizing coe codon, the production of Coe protein was significantly increased. This vaccine expression system provided a new direction for the heterologous expression of vaccine protein in fungi host.