Preliminary Construction Of Rescue System For Goose-host Paramyxovirus Strain NA-1

Goose-host paramyxovirus was founded in our country in 1997 .It was highly contagious and had caused enormous losses on the goose industry in our country.Traditional Newcastle disease virus Lasota vaccine should not have a good immune protection and there is not attenuated virus vaccines for waterfowl of this virus by now.The cycle to advanced development new vaccine used traditional method was too long,so people need to apply new method to develope new type vaccine for waterfowl. APMV belong to single-strand negative strand RNA virus, gene typeâ… . RNA virus genome is more vulnerabale to degradation in vitro,so the new technological for the new type of RNA virus vaccines developes slowly. 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 and provide the technical foundation for the new type of virus vaccine.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.Researchers can achieve changed the virus genome by changing its cDNA.Researchers abroad established the rescuing system of NDV in 1999 and gained many technological achievements such as NDV live viral vector recombinant vaccine. This experiment aimed at establishing the goose-hot paramyxovirus virus rescuing system and to lay the foundation for artificially weak toxical vaccine.The establishment of the rescuing system for virus mainly include the virus genome cDNA synthesis as well as full-length fragment of genomic clone and connection, Plasmid-assisted Construction ,Sequencing of the cloned genome Cotransfected sensitive cell to rescue virus and the identification and application of the new virus .etc. In this study,the following job were done focusing the above contents:1 the structural gene amplified of Goose-hot paramyxovirus strain NA-1The structure gen was amplified using RT-PCR method and its primers were designed according to the Goose-hot paramyxovirus strain NA-1 record on GenBank. The objective gen fragment was reclaimed from agarose gel and connected to T vector. The connection product was transformed into E. coli and then sequensed by biotechnology company. To be eventually the correct plasmids were obtained and were named as: T-G1,T-G2,T-G3,T-G4,T-G5 and T-G1.2 The amplification of Goose-host Paramyxovirus strain NA-1 terminal endsGoose-host paramyxovirus strain NA-1 was separated and purified in our laboratory. Complete RNA genome of virus was extracted and the genome nucleotide sequence terminal ends were amplified by RACE method. The extracted virus genome RNA was connected with a section of artifitial oligonucleotide using RNA ligase. This section of artifitial oligonucleotide was designed according to primer which designed by Peeters B P H and its complementary sequence regard as primer to amplify the first chain of cDNA in reverse transcription. The 3'terminal end was amplified using this pimer and a specific pimer. Genome 5 'end was amplified using cRACE method.3 The construction of Goose-hot paramyxovirus strain NA-1 full-length cDNA and the rescuing of the virusThe 6 fragments which were obtained in the experiment 1 were connected to a high stringency vector pCI-neo.After connected the structure gen was connected with a transcription vector which contains terminal ends and so constructed the plasmid PVAXâ… -G6-1 which contained the full-length cDNA. PVAXâ… -G6-1 and the pCI-NP, pCI-P and pCI-L plasmid which constructure in our laboratory co-transfected the BHK cell line which could express RNA polymerase to rescue virus. After several transfection we did not get the virus. Broke the cell line and then have an inoculation to chick embryo. Passaged 6 generations the hemagglutinating of allantoic fluid was: 20,21,23,23,24,22 ,and can be inhibited by NDV antibodies,so the rescue systemhas been initially established.