Analysis In Genetic Variation Of Isolated NDV And Screening Of McAb 1E5's Mimic Antigen Epitopes

New Castle disease is an acute, highly contagious disease caused by New Castle virus, which causes high rate of infection and death and great economic losse to poultry industry.The hemagglutinin-neuraminidase on the exterior of NDV is main immune sheltered antigen, which have relationship with virulence and nosogenesis.The isolate was named SHY06, isolated from the hydrosalpinx fluid of layer. SHY06 was determined as a virulent strain with MDT of 52h, ICPI of 2.0 after plaque-purification. The amino acid sequences of the fusion protein cleavage site in the isolate was 112-RRQKRF-117, which is a typical sequence of velogenic strains and is in agreement with the results of in-vivo pathogenicity tests. For genotyping, a phylogenetic tree based on nucleotides 1-374bp of the F gene was constructed using SHY06 and 19 other representive NDV sequences from GenBank. Phlogenetic analysis showed the isolate was of the genotypeⅦvirus. The homology analysis of the amino acid sequences of F and HN gene campared to reference strains from GenBank indicated that the F protein amino acid sequences has homologies of 97.5%～99.5% with genotypesⅦstrains, 91.7%～92.4% with genotypeⅨstrains F48E9, JS9701 and SBD02, 89.2 % with genotypeⅡstrain LaSota. While the amino acid sequences of HN gene were compared , SHY06 demonstrated higher homologies of 92.1～99.7% with strains which were isolated in recent years, but lower homologies of 87.6% and 89.9% with LaSota and F48E9, respectly.The monoclonal antibodies 1E5 and C3-B7 are directed against HN gene of NDV, they separately belong to IgG2a and IgG1 and have HI function. They were purified by using affinity chromatography with HiTrap protein-A. After identified the contents in different stages, the result showed that the first 1~2mL Elution Buffer had more protein. The purified McAbs were determined by SDS-PAGE respectively. The results indicated that each purified McAb took on two bands in SDS-PAGE electrophoresis.We screened the simulation epitide of McAb 1E5 after got the purified monoclonal antibody. To get the epitope site of NDV-HN protein which may be identified with McAb 1E5, monoclonal antibody of NDV-HN protein was used to screen the phage display random 7-peptides. The positive clonies were identified by indirect ELISA and competitive ELISA. 4 groups of 7-peptides sequence were got. These sequences were located at the residue 388~395 aa of NDV-HN protein. The mutual sequence L * */ * PNT is the framework structure.