Construction And Selection Of Recombinant Vaccinia Virus Tiantan Strain With Eigth Genes Combined

Vaccinia virus (WR strain) was successfully used in the expression of herpes virus TKgene for the first time as a vector in1982, receving great concern of the research scholars.However, vaccinia virus Tiantan (VTT) strain of China has independently expressed exogenousgenes in1984, which is used as a vaccine to prevent the emergence of variola virus. As vacciniavirus can be inserted large exogenous genes, it can be used in the construction of multiplecombined vaccines.With the deterioration of environment, more and more people were faced with thechallenge of the disease. Thus, the workloads of vaccine immunization were increased whichresulted with waste of resources and risk of infection. Multiple combined vaccines coulddecrease the number of vaccination, reduce the incidence of side reactions and avoid the tabooamong many vaccines.We analyzed the genome of VTT strain and constructed the multiple candidate vaccineswith vaccinia virus of Tiantan strain containing eight exogenous genes which includes Yellowfever virus E gene, Hantavirus S gene, Crimean-Congo hemorrhagic fever virus M gene,Bacillus anthracis POAX2gene, Ebola hemorrhagic fever virus S gene, Marburg hemorrhagicfever virus G gene, Sin Nombre viral G2genes and Africa O’nyong-nyong virus E2gene. Therecombination of exogenous genes is in the flank gene of TC7L-TK2L and TJ2R. The shuttleplasmids contained the recombinant arm, exogenous gene, the promoter and the marker gene.Under the effect of the liposomes, the plasmids enter into cells that infected with vaccinia virusand then genomic recombinated. Recombinant viruses with fluorescent marker were screened byfluorescence microscope after cytopathic effect. The marker genes were knockouted byCre/Loxp gene recombination system. We obtained multi-recombined vaccines without markergene.The results showed we successfully synthesized two gene fragments which separatelycontained four exogenous genes,one of which contained Yellow fever virus E gene, HantavirusS gene, Crimean-Congo hemorrhagic fever virus M gene, Bacillus anthracis POAX2gene andanother one contained Ebola hemorrhagic fever virus S gene, Marburg hemorrhagic fever virusG gene, Sin Nombre virus G2gene and Africa O’nyong-nyong virus E2gene; The two genefragments were used to construct the shuttle plasmid containing marker genes; finally obtainrecombinant vaccinia virus TianTan strain. The recombinant vaccine virus has a well stability ingenetics which is identified by PCR and posess complete shape watching in the electron microscopy which laid a foundation for the development of multiple recombined vaccines.