| Canine distemper(CD) is an acute and highly contagious disease caused by canine distemper virus(CDV). CDV has a wide range of hosts and infects most of the carnivorous species, including Canidae, the Raccoon family,the large Viverridae family and some Felidae. Animals may have a variety of clinical symptoms after infected by CDV, and become easy to get secondary infection of other pathogens, some wild animals like red panda mortality rate is up to 100%. In recent years, the host range has been expanding as the mutation and evolution of CDV,and it was reported that wild animals like giant panda, red panda,tiger or lion could be infected. At present, The method to prevent CD for pets mainly depends on traditional attenuated vaccine.Although some wild animals pets traditional attenuated vaccines have a role in the control of CDV,there still exist some problems in its safety and immune effects for giant panda, red panda,black hoof skunk and other wild animals.Therefore, in order to protect the wild animals from CD and avoid the virulence reversion of attenuated vaccine,it is necessary to develop a safe and effective engineering vaccine of CD.CDV is a member of the Morbillivirus genus in the Paramyxoviridae family,which has two glycoproteins(Hemagglutinin H and Fusion F Protein) that are major target antigens for eliciting neutralizing antibodies.The replication-defective goatpox virus has host restriction that only cause transient infection for mammals and can be used as a safe vaccine vector.Therefore,it is a good vector to construct CDV H and F genes recombinant vaccine.In this study,H and F gene sequence of CDV in giant panda are modified and synthesized based on the published coding sequences on Gen Bank. By the Liuhe Beijing Hua Da gene company.firstly the gene trnsfer vector p MDTK-PEL was constructed on the basic of vector p MDTK-PEL-EGFP. The H and F gene from CDV was cloned into the transfer vector p MDTK-PEL,respectively,hrough restriction enzyme digestion, H、F Gene Expression Cassettes was inserted into the vector p TK-Eg, we obtained the recombinant transfer vector plasmid p TK-H-Eg and p TK-F-Eg,The recombinant transfer vector plasmid was transfected into BHK cells that had been infected with goatpox virus,respectively, through homologous recombination generation of recombinant ruminants virus H 、F gene goatpox virus.The recombinant goatpox virus respectively containing H、F gene goatpox virus cultured in African green monkey kidney cells(Vero cells) are seleted on the pressure selection by adding Mycophenolic acid(MPA) in the culture,after purifying and the PCR、greenfluorescence test and Western blot identification,demonstrated that the CDV H、F gene had been insertion to goatpox virus and correctly expressed CDV H/F protein in cells, we obtained the recombinant GTPV AV41 name as vp TK-H-Eg、vp TK-F-Eg, respectively. This study lay a firm basic for developing genetic engineering vaccine of CD. |
