Construction And Identification Of The Helper Plasmids For Rescuing Goose-host Paramyxovirus Strain NA-1

Newcastle disease (ND) is a serious avian disease with worldwide distribution that can cause severe economic losses in the poultry indusity.The causative agent of the disease, Newcastle disease virus (NDV) or avian paramyxovirus type 1, is a member of the Paramyxoviridae and has been assigned to the genus Rubulavirus in the subfamily Paramyxovirinae. Avian Paramyxovirus consisted of nine serotypes, APMV 1~9, but APMV-1 only included Newcastle disease virus. There existed tremendous virulence diversity among different hosts in which chicken was the most sensitive, pigeon and parrot also could be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. There were outbreaks of clinical ND in goose flocks in Southern and Eastern China have been reported frequentaly since late 1997.This disease entity has posed a great threat to goose production and other poultry industry and attracted much attention. Uninterruptly expanding of the host infection spectrum about NDV, chicken, pigeon, parrot and some rare birds could be infected,as well as waterfowls. The outbreak of goose-host paramyxovirus disease broken traditionary theory which said waterfowls could not be infected by NDV.Several studying results indicated that the genome of NDV evolved at an integrity level.Goose-host paramyxovirus strain NA-1 was separated and purified in our laboratory. Complete RNA genome of virus was extracted and the genome nucleotide sequence of goose-host paramyxovirus strain ZJ1 in Genbank was refered to designed five pairs of specific primers,and then the NP, P and L gene fragments were amplified by the method of reverse-transcription polymerase china reaction(RT-PCR).The six nucleotides were observed between 1643 and 1644 site corresponding with the other seven NDV strains, such as strains Aus/32, Herts/33, B1, Beaudette C, HB92 V4, LaSota and Ulster.Reverse genetics manipulation has been established in recently years based on cloned cDNA of RNA viruses.It has gradually become a very significant method for the research of molecular virology,especially for the research of virus funcitional genome,construction of chimeric viruses and expresss of heterologous genes. NDV is a single strand negative strand RNA virus.It is possible to rescue the NDV.Infectious virus has been generated from cloned full-length cDNA of newcastle disease virus and three helper plasmids for NP, P and L protein were cotransfection cells.Based on the complete genome sequence of goose paramyxovirus strain NA-1 and then four pairs specificity primers were designed to amplified the open reading frame (ORF) of NP, P and L gene fragment by the method of RT-PCR.Meanwhile, the target gene fragments were reclaimed and purified to insert into eukaryotic expression vector pCI-neo vector to converted into the E.coli DH5α. then the positive clones were identified by enzyme digestion, PCR and sequencing. The recombinant plasmids of pCI-NP, pCI-P and pCI-L were transfected into Vero cells, and the green fluorescence was observed under fluorescent microscope with the method of indirect immunofluorescent test, indicating that the three helper plasmids could successfully expressed. After to enlarge culture the Vero cells,the cells were collected and breakdown by the solution of cell disruption, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that specific protein bands about 53.0ku and 42.0Ku appeared on the gel, Western blotting was performed for identification purpose, the result showed that the bands on the gel were specifically combined with the polyclonal antibodies of Goose-host paramyxovirus strain NA-1, and approved that the expressed product were NP and P protein. In vero cells the special protein could not be detected by the methods of SDS-PAGE and Western-bloting after pCI-L plasmids being transfected. It was may be in that the target nucleotides fragement was too large to express in vitro, or was expressed out very very microcrystalline protein which couldn't be detected in our lab. But the method of RT-PCR was adopted to detecte the expressed mRNA in transfected cells using special primers and it indicated that the plasmids pCI-L had been expressed in vero cells.The full length cDNA of Goose-host paramyxovirus strain NA-1 V protein gene was cloned from the plasmid containing Goose-host paramyxovirus strain NA-1 P gene and a necessary mutation for the expression of the V protein gene was introduced by PCR.The mutant V gene was subcloned into the eukaryotic expression vector pCI-neo vector. The recombinant plasmids of pCI-V were transfected into Vero cells, and the green fluorescence was observed under fluorescent microscope with the method of indirect immunofluorescent test, After to enlarge culture the Vero cells, the cells were collected and breakdown by the solution of cell disruption, SDS-PAGE analysis indicated that specific protein bands about 28.0ku appeared on the gel, Western blotting was performed for identification purpose, the result showed that the band on the gel were specifically combined with the polyclonal antibodies of Goose-host paramyxovirus strain NA-1, and approved that the expressed product was V protein. indicating that the plasmids could successfully expressed.