Construction Of Recombinant Marek’s Disease Virus CVI988/Rispens Transfer Vector Expressing Infectious Bursal Disease Virus-VP2Protein

Infectious bursal disease (IBD) is an acute and highly contagious disease of chickens. IBD makes high economic losses each year. In addition the emerging of very virulent strain will be a threat in preventing IBD infection. Intermediate and weak pathogenic live vaccine is widely needed to prevent IBD.The maternal antibody in commercial chickens impaired the immunity effect of the live vaccine. Virulent virus live vaccines are used in commercial chicken, but they caused more serious bursal pathological lesions and immunosuppression resulting in the emergence of very virulent and variant strain.Domestic and foreign scholars are still trying to use molecular biology and bio-technology methods to develop novel vaccine against IBD. In recent years, these trails and efforts have made a great progress. The field experience has clearly demonstrated the safety and effectiveness of the MDV vaccines as a viral vector. In this study, we used the CVI988vaccine strain as a vector, no-essential gene US2as a gene insertion site and a CMV promoter to construct recombinant MDV transfer vector,and then we preliminarily obtained the recombinant Marek’s disease virus indentified by indirect immunofluoresent assay (IFA)1Construction of a universal transfer vector pUC-VP2-US2ofCVI988strainThe right and left fragments on either side of the MDV CVI988US2gene were amplified by PCR using primers containing appropriate restriction enzyme sites.Both fragments were subsequently cloned into pUC18to obtain pUC-US2with restriction enzyme site Xholl and BglIL The complete IBDV-VP2express cassette under the CMV immediate-early promoter was inserted into the plasmid pUC-US2to create the transfer plasmid pUC-VP2-US2without any reporter gene. The secondary CEFs were transfected with recombinant plasmid pUC-VP2-US2by lipofectamine2000reagent. The indirect immunofluorescence result showed that the plasmid can express IBDV-VP2protein in CEFs, which lay foundation for further transfecting CEFs infected MDV CVI988and obtaining recombinant MDV expressing IBDV-VP2protein.2Transient expression of plasmid pUC-VP2-US2and preliminarily identification of rMDVThe CEFs growth curve was generated by traditional counting of the cell number, and the logarithmic growth phase of CEFs was determined. We transfected pUC-VP2-US2into CEFs by using lipofection method.The effective of some parameters on transfection were optimized,such as time of transfection, the ratio of lipofectamine and plasmid (volume/quality) and cells density and so on.We determined the optimum transfection condition:Seed2×105secondary CEFs per well into24cell culture plate, after24hours the secondary CEFs were transfected with1.5μl lipofectamine and1.0μg plasmid pUC-VP2-US2.The optimum transfection condition improved the possibility of recombinant Marek’s disease virus occured. On the optimal transfection condition, the linearized pUC-VP2-US2was transfected into CEFs infected with MDV-CVI988to make a homologous recombinant virus inside the cells. Analysis results with indirect immunofluorescence assay showed that the recombinant MDV-CVI988expressing IBDV-VP2without any reporter gene was prelimitarily obtained.