Preparation Of Maedi-Visna Virus DNA Vaccine And Its Immunological Research

Maedi-visna virus (MVV) belongs to the lentivirus subfamily of retroviruses, the monocytes and macrophages are the natural target cell in vivo. It integrates into the host cell DNA causing a persistent infection with a tropism mainly for the cells of monocyte-macrophage lineage and distributed in various tissues. The virus causes chronic inflammatory disease in persistently infected sheep, including progessive pneumonitis, mastitis, encephalitis and less commonly, arthritis. MVV was first found in 1920s and spreaded world wide nowadays. MVV can be transmitted to the lambs by suckling colostrums or milk from the infected mother and other sheep by animal repiratory system and digestive system. MVV was first present in China in 1960s. Thus in the absence of effective treatment methods and vaccines for MVV, a new vaccination appears as the most suitable alternative. The study on the gene cloning and its nucleic acid vaccine is a significant way to find the relationship between MVV and other lentivirus stain and protect the sheep from such a severe disease.The gene cloning and sequence analysis of MVV The several main genes were cloned and analyzed to tesify the virμlenc of MVV and the relationship with original strain. According the these genes sequence of MVV(Accession Number: AY101611), 4 pair of primers were designed, and 4 fragments were obtained and sequenced by PCR amplification from the whole viral genome of infected cells extracted by the method of Proteinase K. Restμlts of nucleotide sequence analysis showed that there are high nucleotide homology between the genes of MVV-am and original Amrican strains MVV-85/34, which is about 96.9%. Comparatively, the homology of LTR and pol is low and gag core fragment is high. The nucleotide homology of MVV-am and 85/34 strain compared with MVV-1514 is is highly variable, which is about 90.1%.The protective antigen genes cloning and its sequences analysis The main protective antigen gene gag was cloned and its nucleotide sequences and antigenicity were analyzed for the study on MVV nucleic acid vaccine. DNA fragments of main protective gag gene were amplified from the proviral genome DNA of MVV by PCR using primers designed according to the nucleotide sequence of MVV American strain. The gene was cloned to pGEM-T easy vector and transformed to E.coli, The postive clones were selected and sequenced. The nucleotide sequence analysis showed that the nucleotide of gag of MVV-am shared 98.2% and 92.6% similarity to that of MVV-85/34 strain and MVV-1514 strain respectively, the amino acid homology of Gag compared with MVV-85/34 and MVV-1514 strain is 96.9% and 90.2% respectively. The analysis of antigenicity of gag core fragment with DNASTAR showed that both of them have high antigenicity and can be used to the studies of nucleic acid vaccine.Construction of the eukaryotic plasmid pcDNA5/TO-gag The MVV nucleic acid vaccine...