Cloning And Expression Of The Fusion Gene Of Newcastle Disease Virus Strain F48E8 By Recombinant Fowlpox Virus And Characteristic Of Its Immunity

In this paper, the Fusion protein of NDV strain F4SES was amplied from the plasmid PE1/E2-3 by polymerase chain reaction(PCR). In order to express the F gene of NDV F48E8 in the expression system of fowlpox virus(FPV),we construct a FPV transfer vector including NDV F gene.The open reading frame (ORF) of F gene was cloned into the FPV tmnsfect vector pEFgptl2S downstream of S promoter. The positive clone was selected by anti-kanamycin and demonstrated by restriction endonuclease analysis. Chicken embryo fibroblast(CEF) cell which were infected with FPV 282E4 for 2h were transfected with the complex of pEFNDF plasmid and calcium phosphorus .Recombinant FPV was screened by using E.coli guanine phosphoribasyl transferase(gpt) gene located downstreame of the S promoter of pEFNDF plasmid and the selective drug of mycophenoic acid added into the medium.We can get recombinant FPV stably expressing NDV fusion protein through many times sclection.The rFPV including the F gene of NDV F48E8 was confirmed by PCR and DNA dot hybridization assay.The expression of NDV F gene in rFPV was also confirmed by SDS-PAGE and Western blotting assay.After that, the characteristic of its immunity was detected by vacoinated the II day-old chickens with the normal dose of rFPV, NDV LV vaccine,rFPV+IV and FPV respectively.The blood samples were collected to test the antibody titers against F protein by HI and ELISA. and NT antibody against FPV The chickens were challenged by virulent NDV F48E8 4, strain at 34 days old. My study proves the rFPV expressing NDV F gene can effectively protect chickens against chanllenge of virulent NDV The antibody anti-F protein was detected in the serum of chickens vaccinated with rFPV using ELISA . The extent of protection afforded by rFPV is 80% ,all chickens vaccinated by rFPV-I-1V can be protected.We can also find anti-FPV antibody in the serum of chickens vaccinated by rFPV...