| Chick embryos were inoculated with NDV F43E9 strain and the pathomorphological changes of the sick embryos were examined. The results showed all the sick embryos were hypoevolutism, weak, flush and there were many hemorrhagic spot in the surfaces of the sick body, Hyperemia, congestion, edema, hemorrhage and parenchymatous degeneration were found in stomach, intestine,liver, brain, kidney, heart and skeletal muscles, the NDV particle was observed though electron microscope in the purificated allantoic liquid of sick embryos. Two pairs of oligonucleotid primers PU1/PLI and PU2/PL2 were designed according to the published P protein and M protein gene sequences of NDV.Two fragment of 1.1kb and 1.6kb were amplified by RT-PCR from RNA of F48E9 strain,and cloned into pMD18-T vector respectively. The recombinant plasmids were digested by restriction enzyme EcoRI, HindIII and MseI,and subcloned into pMD18-T vector again, as a result, we obtained complete P protein gene clone of NDV F48E9 strain. It was found that the P has 87% and 91% homologies with La sota in nudeotide and amine acide level ,respectively.It was concluded that strain F48E9 is genetically closer to strain La sota There were four epitopes across the squence of the P,they are 20-28,113-112,190-200 and 220-230 amine acid residues. The recombinant plasmid pMD1S-T-P was digested with EcoRI/HindIII and subcloned into PFASTBAC1 digested with EcoRI/HindIII. The 時esulting recombinant PFASTBACI-P contains P gene at downstream of the polybedrine gene initiatioin codon. The recombinant PFASTBAC1-P was transposed into the DH1OBAc competent cell and the recombinant bacmid DNA was isolated and subjected to identification by PeR. The recombinant baculoviruses were obtained by transfection of sf9 cells with the the recombinant bacmid DNA and CELLFECTIN. The infected insect cells were collected at different time post-infection, analysed by SDS-PAGE. The results showed that the expressed product had a molecular mass of approximately 56kD. |
