Construction And Assemblage Of Recombinant Adenoviral Vector Carrying Muscovy Duck Reovirus σC Encoding Gene

Muscovy duck reovirus(MDRV)disease is one kind of high incidence,high mortality muscovy duck infective diseases.It has caused serious economic losses in the muscovy duck industry since it happened,and became one of the main epidemic diseases which restrict the healthy development of the muscovy duck industry in our country.Presently,the research of MDRV vaccine which is reported at home and abroad is only the MDRV attenuated vaccine screened by the Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences.This attenuated live vaccine has a good rate of protection,and has played an important role in the effective control of the muscovy duck reovirus disease.But the attenuated vaccine also shows some safety problems such as sheding virus and virulence reversion.Therefore,it is necessary to develop a more security and effective vaccine.As for the purpose of this,Muscovy duck reovirusσC encoding gene recombinant adenoviral vector was constructed,and the recombinant adenovirus was assembled in the AD293 cells.This laid a technical foundation for the research of the MDRVσC encoding gene recombinant adenovirus vaccine.According to the nucleotide sequence of MDRV MW9710σC encoding gene,we designed a pair of primers.The objective fragment was amplified from theσC encoding gene recombinant T vector plasmid,then theσC encoding gene fragment adding Kpn I and Not I restriction site in the both ends was constructed.After digested by Kpn I and Not I enzyme,the objective fragment was ligated with the pShuttle-CMV shuttle vector via the method of sticky end ligation,and pShuttle-CMV-σC recombinant vector carryingσC encoding gene was constructed.Then the pShuttle-CMV-σC vector prelinearized by Pme I was homologously recombinated with the pAdEasy-1 skeleton vector via electroporation transfoming the BJ5183 competence cells,and the recombinant adenoviral vector pAdCMV-σC was constructed.After linearized by Pac I,the pAdCMV-σC was transfected into AD293 cells.In the 7th day post transfection,the cells showed evidence of cytopathic effect(CPE).This indicated that the recombinant adenovirus has already assembled in AD293 cells.Recombinant virus was serially passaged and identified by PCR,and the result showed that theσC encoding gene stably existed in the genome of the recombinant adenovirus.The result of limited dilution method showed the TCID₅₀of the recombinant virus was 10⁹TCID₅₀/ml.