Phylogenetic Analysis And Nucleocapsid Protein Expression In Insect Cells Of Peste Des Petits Ruminants Epidemic Strains Isolated From Guangdong Province

Peste des petits ruminants( PPR) is a highly infectious diseases caused by the peste des petits ruminants virus( PPRV). The viral major harm goats, sheep and other small ruminants. With sudden illness, high fever, Aphthous, nasal mucus, diarrhea and other major symptom, on account of its high morbidity and mortality, Spread quickly, by the World Organisation for Animal Health(OIE) as Class A one of potent major infectious diseases. Peste des petits ruminants virus was first discovered in July 2007 in China’s Tibet, Peste des petits ruminants viruse was heavy outbreak in 2013 in Xin Jiang province, causing large quantities of goat, sheep death, mading farmers the uge losses.Then most prevalent and spread in the country. Of which more than 20 provinces,municipalities. Local authorities also pay attention to the harm of the virus, and actively take measures to prevent it. In the spring of 2014, the author first detected Peste des petits ruminants virus of a sheep farm in Guangdong Province, and named CH / GDDG / 2014 strain. The study complete genome characterization, N antigen gene activity of peste des petits ruminants. it helped to understand the molecular epidemiology of the virus, but also laid the foundation for the diagnosis of peste des petits ruminants.The study results are as follows:1.Using RT-PCR, full-length sequence of peste des petits ruminants virus were obtained from the genome of this PPRV strain by eleven over-lapping c DNA fragments amplified,and the strain named CH/GDDG/2014. Then, we analyzed the homology in nucleotide and among the 23 reference strains PPRV included in Gen Bank and constructed systematical tree of evolution. The CH/GDDG/2014 genome comprised15,954 nucleotides(six nucleotides more than classical PPRVs identified before 2013,but complying with the rule-of-six) with six open reading frames(ORFs) encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. According to the consequence, the homology in nucleotide from reference strain PPRV samples was from 87.2% to 99.8%, The whole genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus(99.8%) to China/XJYL/2013 and the least consensus(87.2%) to KN5/2011.Thus, the PPRV samples from domestic in recent 2 years are in the same branch of systematical evolution, and with aborad strains in different branches. Indicates that the current domestic PPRV are in closer relationship, and mainly in lineage IV epidemic.2.This study successfully constructed N gene of recombinant shuttle plasmid p Fast Bac-PPRV-N of Peste des petits ruminants, after properly sequenced, transformed into DH10 Bac Escherichia coli cells, obtained recombinant baculovirus bacmid Bacmid-PPRV-N through the blue-white screening after transfection sit, transfected Sf9 cells of grow well, got recombinant baculovirus. The identification Bands N protein expression consistent with the expected through SDS-PAGE and WB.