The Construction Of Plasmid Cloning System Of New Castle Virus JL02/2000 And The Helper System

Newcastle disease(ND) is a highly pathogenic viral disease of avian species, which is economically significant as a result of the huge mortality and morbidity. The development of reverse genetics systems for manipulation and study of RNA virus genomes have provided platforms for designing and optimizing viral mutants for vaccine research, which makes great significance to study biological characteristics. While its auxiliary plasmid replication must be transfected more than two plamids at the same time, and then, under the regulation of T7 RNA polymerase, RNP ribonucleoprotein complex is formed after transcription, translation, and the final packaging of virus genome. After that, the virus begins to replicate to produce next-generation virus. This experiment built a stable expression of recombinant vaccinia virus used to provide a T7 RNA polymerase and built the three auxiliary plasmid pCI-L, pCI-NP and pCI-P, which was involved in the formation RNP complex. Finally, we constructed the whole genome of NDV eukaryotic expression plasmid. Based on the method of SOE-PCR, we built the whole genome of starting gene ND1. In order to successfully clone ND1 gene, we built plasmid pCI-T. Besides, we built transfer plasmid pBSK-0-1-2-3 and eukaryotic expression plasmid pBR322-ND5. Thus we completed the Newcastle disease JL02/2000 strains of reverse genetic operation platform and the genome of cloning.