Development And Immune Potency Evaluation Of Recombinant Saccharomyces Cerevisiae Expressed Newcastle Disease Virus Proteins

Newcastle Disease Virus(NDV)is a poultry virus that first appeared in Newcastle,England in 1927.NDV mainly damages the digestive system and respiratory system of sick flow,and even causes death.It is although one of the pathogens that seriously threatens the global poultry industry.Rational designed and incubated of vaccines is the main method of prevention and control of ND epidemics.Most of the strains used in existing NDV vaccines are constructed by the strains discovered in the 1950 s,and there is a large genetic gap between the strains that currently cause the Newcastle disease epidemic.Therefore,it is necessary to develop new Newcastle disease vaccine.In this study,the recombinant strain of Saccharomyces cerevisiae expressing NDV F and HN truncated proteins was constructed.Then the efficacy of the recombinant strain was assessed in chicken by oral administration.The main results were listed as follows:1.Assembly of NDV F and HN transcription unit: Based on the predicted B cell epitopes using amino acide sequence,and combine the research results of others,we selected 241 bp to 888 bp of the F gene and the 25 bp to 528 bp of the HN gene.Multiple sequence alignment and analysis of NDV F and HN encoding genes were carried out in bioinformatics.The truncated protein encoding sequence were amplified and inserted into linear transcription unti POT.The F/HN-TU carring NDV protein was fully assembled.2.Construction of F/HN recombinant yeast: With the technology of Yeast Fab Assembly,the transcription unit is connected to the homology arm and Leu nutritional selection marker in series through the different characteristics of type II endonuclease.and F/HN-TU was transfermed into yeast cell by classical Li Ac method.The transformers were identified by PCR,Western blot and immunofluorescence.The results indicated that F and HN proteins expressed and exhibited on the surface of yeast cell.Through the growth curve detection,combined with Western Blot gray analysis results,it was confirmed that the recombinant protein had the highest expression level at 36 hours,compared with the negative control group,the expression of exogenous proteins F and HN had little effect on the growth of the host yeast.3.NDV F/HN recombinant yeast induced immunogenicity in fowl following oral administration.Fowls were randomly divided into two groups.ELISA was performed and the results indicated that Ig G content in the immunized serum was significantly higher than that of the negative serum,which provided an effective strategy for the immune control of porcine NDV.