Construction Of Recombinant Pseudorabies Virus Expressing Both HA And NA Gene Of A Swine Influenza Virus H1N1

Influenza is a common respiratory disease in pigs,and it is a acute,feverish and infectious disease.Swine influenza virus(SIV) especially H1N1 and H3N2 subtype SIV is the prevalent pathogens of SI.Swine is the only animal which can be infected by either avian or human original influenza viruses,and can be considered as the intermediate host for the process of genetic reassortments between viruses of different hosts.Generate new strains of influenza virus.Vaccination against SIV can reduce economic lose and play a role in food safety and public health.1.Construction of an expression vector of two-colour flurescence depending on IRESThe recombinant plasmid pIE-EGFP/DsRed,including two expression cassette for both EGFP and DsRed gene,and pIE-EGFP/IRES/DsRed,including IRES between GEFP and DsRed gene,were generated.Then the both plasmids and genome of pseudorabies virus TK^-/gE^-/LacZ⁺ were co-transfected into confluent swine kidney cells(IBRS-2) respectively,and 24 h after transfected a fluorescent microscope is employed for observation.But,the upsteam gene of EGFP,expressed higher than downsteam gene of DsRed,in the same group.The both EGFP and DsRed can be observed in the two groups of cells.And,The EGFP and DsRed in the pIE-EGFP/IRES/DsRed group expressed obviously higher than the pIE-EGFP/DsRed group's.Therefore,the method,in which IRES connected HA and NA gene,was chosen.2.Construction of recombinant pseudorabies virus expressing both HA and NA gene of a Swine Infuenza VirusBased on the universal transferring vector pIECMV and the plasmid pIRES,the recombinant plasmid pIE-HA/IRES/NA,including both HA and NA gene of a Swine Influenza Virus Isolate A/Swine/TianJin/01/2004(H1N1),was generated.Then plasmid pIE-HA/IRES/NA and genome of pseudorabies virus TK^-/gE^-/LacZ⁺ were co-transfected into confluent swine kidney cells(IBRS-2).Pathologic cell suspension was harvested and used for plague-forming test and consequent PCR screening.Finally the expected recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ was achieved.The present virus obtained was demonstrated the designed homologous recombination by westhen blot analysis and indirect immunofluorescence.3.The Study on biological property of the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺Results the target segment could be detected at eath serial passage form 1 to 10 of the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ on the PK-15 by PCR.The TCID₅₀ of PRV Ea TK^-/gE^-/LacZ⁺ and the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ were determined after the third serial passage on the PK-15.The TCID₅₀ of the different virus on the PK-15 were TK^-/gE^-/HA⁺/IRES⁺/NA⁺ 10^-5.5,TK^-/gE^-/LacZ⁺ 10^-7.5.Study on biological property displayed the recombinant virus has a fine genetic stability,however the ability to propagate in PK-15 cells decreased a little.4.Safety test of the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ in BALB/c mice6-8 week old BALB/c mice were immunized by the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺(n=6).It is normal for the mice during the 14 days observation. It was safe to BALB/c mice.5.Humoral immune of the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ in BALB/c mice6-8 week old BALB/c mice were immunized by the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺(n=6).The serum were collected on 14 and 28 days after immunization and the immunogenicity of the recombinant virus TK^-/gE^-/HA⁺/IRES⁺/NA⁺ were tesed by enzyme linked immunosorbent assay(ELISA) using whole-virion SIV H1N1 as the coating antigen.The immunity study indicated that it could elicit antibody against H1N1 influenza virus which was detected by ELISA.