Molecular Characteristics Of The Major Glycoproteins Of The Novel Pseudorabies Viruses And The Construction Of Transfer Plasmid For GE Gene Deletion

Pseudorabies virus（PRV） is a member of the family Herpersviridae, subfamily Alphaherpesvirinae,with a broad range of animal infections. It is well known that latent infection with PRV easily occurs in its natural reservoir, pigs. This causes serious economic losses in the swine industry worldwide. Before 2011, the PRV was controlled well because of the Bartha-K61 vaccine, but in 2011, there was a sudden outbreak of domestic epidemic the Swine Pseudorabies and it had proved that the classic Bartha-K61 vaccine can’t provide efficient protection,and it made the domestic swine industry under threat. This study analyzed the glycoprotein B、C、D、E gene sequence of PRV novel epidemic virus strain in this study; it analyzed the genetic difference between the novel epidemic PRV strain and Bartha strain, which provides theory basis for further clarification of no efficiency of Bartha-K61 vaccine. After that,a new plasmid was constructed to remove gE gene of novel epidemic PRV strain.And the new recombinant virus is helpful to study the new vaccine for novel epidemic PRV strain.The contents of the detailed research are as follows:1.The isolation and identification of novel epidemic PRV strainThe brain tissue from a pig farm in An Hui was delected by PCR, and It’s a positive result for gE gene. The BHK-21 cell was inoculated by mashed brain tissue, As for diseased cells, It’s also a positive result from the PRV gE gene PCR detection. After that, the virus was isolated by plaque purification.,named PRV AH-China-2013. Then, the Kun Ming mice was inoculated by PRV AH-China-2013, and the Pathogenicity was evaluated by measuring LD₅₀, the result show that the LD₅₀ is 10^4.15TCID₅₀.2.The analysis of novel epidemic PRV strain glycoprotein molecular features。According to the published gene sequence from novel epidemic PRV strains in China, the primers of gene g B、g C、g D、gE were designed,and all of the genes which were amplified from PCR were sequenced, and the phylogenetic trees of g C and gE were made,and all of the sequences were analyzed by camparing with different classical PRV strains.The result showed that all the domestic PRV strains were in a same branch except the stran which isolated in 2008,and the strains which isolated after 2011 showed the genetic affinity. It proves that PRV AH-China-2013 strain belongs to the novel epidemic PRV strains.In addition,camparing with the classical PRV strains from abroad,the domestic PRV stains isolated after 2011,the main glycoprotein genes of domestic PRV strains showed obvious genetic mutation,and some of the mutations in the critical area of genes, and the mutations maybe is the reason for the sudden outbreak of domestic epidemic the Swine Pseudorabies.3. The plasmid construction for gE-delected recombinant PRV.Accoding to the published PRV strain ZJ01, a pair of primers were designed for two genes respectively located in front and back of gE gene, they are the homologous arms for homologous recombination named LA and RA. Meanwhile, another primer was designed for fluorescent protein from the published plasmid p EGFP-N1, named EGFP. After that, connecting LA、RA and EGFP into vector PMD-18 T with the ways of enzyme digestion、connection and conversion. Then the cell transfection for fluorescent protein gene expression from new plasmidIn this study, It was analyzed the genetic difference between the novel epidemic PRV strain and the other classical strains, which provides theory basis for further clarification of no efficiency of Bartha-K61 vaccine and a new plasmid was constructed which provides basis for PRV homologous recombination.