Construction Of The Recombinant NDV Expression VP2Gene Of Wibdv By IRES

In pace with the reverse genetics technology, NDV has been developed as a live viral vectorfor expression of exogenous protective antigens. There are so many bivalent vaccines based onNDV live viral vector, such as NDV expressing VP2gene from IBDV, S gene from IBV and HAgene from avian influenza virus. However, Majority of these vaccines are unable to achievedesired immunological efficacy because of low expression level of protective pathogen antigens.Therefore, the expression level of exogenous protective immunity gene in recombinant NDV is low.It limits the use of recombinant NDV vaccines. Improvement of the expression level of exogenousgene is crucial for the development and application of genetically engineered live vector vaccines.VP2is the major structural component and the host protective antigen containing the viralneutralizing epitopes, which is responsible for the production of neutralizing antibodies. Toenhance the expression of exogenous gene in recombinant NDV, we successfully constructed therecombinant NDV (rClone30-VP2-IRES-VP2) containing double VP2genes combined with theinternal ribosome entry sites (IRES) sequence. Another recombinant virus (rClone30-VP2) wasconstructed as control containing single VP2gene. Hpa I、BamH I and Sac II were introduced tothe5’ end and3’ end of the VP2gene; BamH I、EcoR I、Mlu I and Sac II were introduced to the5’end and3’ end of IRES gene; EcoR I、BamH I and Mlu I were introduced to the5’ end and3’ end ofthe second VP2gene by PCR. The VP2gene、IRES gene and the second VP2gene were cloned topMD18-T simple vector. IRES gene was cloned into the3’end of VP2gene by BamH I and Sac IIrestriction enzyme sites. The second VP2gene was cloned into the3’ end of IRES gene by EcoR Iand Mlu I restriction enzyme sites in turn. The recombinant plasmid named pMD-VP2-IRES-VP2.The VP2-IRES-VP2fragment was inserted into the NDV full-length cDNA (pClone30), and therecombinant plasmid was named pClone30-VP2-IRES-VP2. Then, pClone30-VP2-IRES-VP2digested by BamH I, the digested fragment was named pClone30-VP2by self-ligation. The twoplasmids were separately cotransfected with helper plasmids expressing nucleoprotein、phosphoprotein and large protein into BHK-21cell which stably expressing T7RNA polymeraseto rescue the viruses. The rescued virus was first inoculated into embryo eggs and the allantoicfluid was harvested for the follow-up experiments, the rescued viruses were respectively namedrClone30-VP2-IRES-VP2and rClone30-VP2. The growth rate of rClone30、rClone30-VP2and rClone30-VP2-IRES-VP2viruses in embryoeggs was similar, which indicated that the insertion of exogenous gene did not affect the viruscharacter. Expression of VP2protein in infected DF-1cells was confirmed by indirectimmunofluorescence assay (IFA). Real time-PCR was used to examine the differential transcriptlevels of VP2gene.The transcript level of double VP2genes in show significant higher than singleVP2gene in rClone30-VP2. The VP2mRNA transcript level of rClone30-VP2-IRES-VP2is1.6times than rClone30-VP2, when the recombinant NDV infected HepG2cells at a MOI of0.001.In conclusion, we construct the recombinant NDV expressing double genes by using IRES,and prove the double genes have a higher expression than single gene. The study provides a newapproach for improving the expression of exogenous genes in recombinant NDV. The result laidthe foundation for the development of the NDV-IBDV bivalent live vector vaccine and providedthe experimental basis for evaluation of the vaccine immunization trials in vivo.