Construction And The Immune Effect Evaluation Of Recombinant Adenovirus Expressing HN Gene Of NDV And GD Gene Of ILTV

Newcastle disease?ND?is an acute and highly contagious infectious viral diseases of chickens caused by Newcastle disease virus?NDV?,which is a highly contagious avian disease with worldwide distribution.As one of the major immunogenic protective protein of NDV,Hemagglutinin-neuraminidase?HN?has become one of the preferred target genes to study the genetic engineering vaccine of Newcastle disease.Infectious laryngotracheitis?ILT?is a highly contagious acute respiratory disease of chickens caused by Infectious laryngotracheitis virus?ILTV?.A large number of researches have shown that the gD,which is exists on the surface of the virus particles not only play a main role in the virus infection but also is the most highly conserved herpesvirus glycoprotein.it is the main protective antigen in ILTV replication and induction of the immune response in the host.It also has been shown to be essential for involved in crucial steps during virus infection,in particular attachment to and penetration into target cells and direct cell-to-cell spread.It can induce high titer cellular and humoral immune respone,and induces a more consistent and stronger cellular immune response than g B and g C.1.Construction and identification of recombinant adenovirus expressing HN and gD geneIn this research,we amplified target genes using RT-PCR and PCR respectively,then cloned the target genes into pMD19-T vector by T-A cloning,named pMD-gD and pMD-HN.After identified by digestion and sequencing,we used T4 ligase to link the target genes with pDC₃₁₅-EGFP,and obtained recombinant adenovirus shuttle plasmid pDC₃₁₅-HN-EGFP ?pDC₃₁₅-HN-gD-EGFP and pDC₃₁₅-HN-gD-EGFP respectively.HEK293 cells were cotransfected with the constructed recombinant shuttle plasmid and pBH by lipofection.After tested by western blot,we constructed recombinant adenoviruses expressing gD gene and HN gene.We purified by Vivapure AdenoPACK and determined with TCID₅₀ method,and the titers of pBH-gD-EGFP ? pBH-HN-EGFP ? pBH-HN-gD-EGFP ? pBH-EGFP were10^10.5TCID₅₀/mL ? 107.875TCID₅₀/mL ? 10^10.5 TCID₅₀/mL and 1011.25 TCID₅₀/m L,which is enough for follow-up animal experiment.2.The immune effect evaluation of recombinant adenovirus expressing HN and gD gene after NDV challengeThe Immunization and challenge test was performed on PBS group,pBH-EGFP,NDV?Lasota?vaccine,recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group.The results showed that the recombinant adenovirus vaccine had no obvious inhibitory effect on the growth of SPF chickens;The results of antibody level showed that there was no significant difference between the NDV?Lasota?vaccine and the recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group?p> 0.05?,and there was significant difference between PBS group and pBH-EGFP group?p<0.05?;The result of lymphocytokine test showed that NDV?Lasota?vaccine strain and recombinant adenovirus group could induce the proliferation of CD3⁺,CD4⁺,CD8⁺and Bu-1a⁺cells.Indicating that the recombinant adenovirus can induce humoral immunity and cellular immunity.3.The immune effect evaluation of recombinant adenovirus expressing HN and gD gene after ILTV challengeThe Immunization and challenge test was performed on PBS group,pBH-EGFP,ILTV attenuated vaccine K317 strain,recombinant adenovirus pBH-gD-EGFP group and pBH-HN-gD-EGFP group.The results showed that the recombinant adenovirus vaccine had no obvious inhibitory effect on the growth of SPF chickens;The results of antibody level showed that there was no significant difference between the K317 strain and the recombinant adenovirus pBH-gD-EGFP group and pBH-HN-gD-EGFP group?p> 0.05?,and there was significant difference between PBS group and pBH-EGFP group?p <0.05?;The result of lymphocytokine test showed that ILTV vaccine K317 strain and recombinant adenovirus group could induce the proliferation of CD3⁺,CD4⁺,CD8⁺⁺and Bu-1a⁺cells.Indicating that the recombinant adenovirus can induce humoral immunity and cellular immunity.In summary,the concluded of this study was to construct the recombinant adenovirus expressing HN and gD gene,and illuminates its growth characteristic and genetic stability,which lay the foundation for research on bivalent genetic vector vaccine of Newcastle disease and infectious laryngotracheitis.