Cloning And Sequence Analysis Of Fusion Protein Gene Of Newcastle Disease Virus Changli Strain And Its Expression In Recombinant Baculovirus

NDV Changli strain was isolated and identified, the biological properties of it was studied. Mean death time(MDT) of chicken embryo and intracerabral pathogenicity index(ICPI) of 1-day-old chicken revealed that NDV Changli strain was mesogenic.The partial F gene of NDV Changli strain was amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was cloned into plasmid and sequenced. The result indicated that the partial F gene of NDV Changli strain was 813bp ,encoding 261 amino acids. There were 2 glycosylation and 4 cysteine residues,The 81 3bp nucleotide and deduced amino acid sequence (261 aa) of NDV Changli strain was compared with published NDV F genes (13 strains) in the same region. Their homology in nucleotide sequence was between 84.1% and 97.5%, 85.8% and 93.9% in deduced amino acid sequence. The phylogenetic analysis of nucleotide sequences of F genes showed that NDV Changli strain and NDV Siping strain was in the same subgroup.The amino acid sequence of the cleavage site region (amino acid residues 11 2~ 11 7aa) of the F protein of Changli strain matched to that of all virulent strains , so it was confinned to be virulent strain.The same region of NDV Siping strain F gene was substituded by the cloned partial F gene of Changli strain, the recombinant F gene was put into pBluescript II and sequenced. The result indicated that the recombinant F gene was I 754bp. containing a entire Opening Reading Frame and encoding 553 amino acids. The deduced amino acid sequence from the recombinant F gene included 3 hydrophilicity plot, this is similar to Siping strain.The recombinant F gene was inserted into the vector pFastBac I ,then transformed into E.coli DH1O Bac including bacmid and helper plasmid .Transposition was carried out and recombinant bacmid was constructed in the E.coli. The recombinant bacmid was transfected into sf-9 cells and the recombinant F protein was expressed. The results of SDS-PAGE and Western-42blot showed that the molecular weight of the expressed recombinant F protein was 63KD.Chickens were immunized with the recombinant F protein and the antibody against F protein was found in chickens?serum by ELJSA. The antibody level can protect chickens against virulent NDV.