The Detections Of Exogenous ALVs From The Live Vaccines And The Constructions Of The Ubiquitin-mediated ALV-J Multigenes Co-expressed DNA Vaccines

Avian leukosis(AL)is caused by avian leukosis virus(ALV)that belonging to retrovirus families,a retrovirus genus,and subgroup J ALV is most common and causes serious threat to the poultry industry.At present,the most effective mean for ALV-J prevention and control is the eradication of the breeders.In order to better serve the eradication program for the industry,the detections of the exogenous ALVs on the live vaccine,which are commonly used in the breeder farms and the constructions of the ubiquitin-mediated ALV-J multigenes co-expressed DNA vaccines,were done as described in the followings.Firstly,totally 45 samples of the commonly used live vaccines,consisting 11 different kinds of the commercial products produced by 14 vaccine companies,provided by four breeder farms in Guangxi and Guangdong were detected for the exogenous ALVs by both the direct PCR and the virus isolation with DF1 cell culture.ALV-J was detected in two of the live vaccine products.The viral genome sequencing and analysis of these two ALV-J isolates were done,97.5%of nucleotide sequences homology was found between the two strains,and have the closest phylogenetic relationship with the strain JS09GY3 which was isolate from a bird experienced clinical AL in 2009 from Jiangsu and nucleotide sequences homology were 95.5%.Secondly,the ubiquitin gene,the gp85,P27 and P10 genes of ALV-J HG03 strain were amplified by PCR with various restriction sites in the PCR products,and then the fragments were ligated to the eukaryotic expression vector pVAX1.Five recombinant plasmids pVAXl-Ubl-gp85,/pVAX1-Ub1-gp85-P27,pVAX1-Ub1-gp85-P10,pVAX1-Ub2-P27-P10 and pVAXl-Ubl-gp85-P27-P10 were successfully constructed,and then were transfected respectively onto the DF1 cells.At 12,24,48,72 hours of post-tracfection the cell cultures were detected by using indirect immunofluorescence assay(IFA)against the expressed target genes with a gp85-specific monoclonal antibody and chicken positive sera against ALV-A/B and ALV-J subtypes,positive results were obtained and that meant the ALV-J genes carried by the constructed recombinant plasmids were successfully expressed in the DF1 cells.The results of the study demonstrated that the exogenous ALV-J was detected in the commonly current used commercial live vaccines;The ubiquitin-mediated ALV-J multigenes expressed DNA vaccines were successfully constructed and expressed in DF1 cells.