Avian Infectious Bronchitis Virus N Gene Vaccine Mediated By Attenuated Salmonella Typhimurium And Study On Immunogenicity

Infectious bronchitis virus(IBV)causes an acute and highly contagious infectious disease in chicken. IBV is the largest RNA virus of coronal virus .Nucleoprotein (N protein)is reserved and compose internal nuclebcapsid which plays an important role in cellular immunity and humoral immunity.Attenuated Salmonella typhimurium X4550 is the preferred strain of balanced lethal host-vector system strains which can be widely used as vaccine vector for oral immunization to carry heterologous antigen of immunoreaction into zooblast and duplicated to express exogenous antigens. Attenuated Salmonella typhimurium X4550 is safe due to their cya and crp of virulence gene were knock-out and used balanced lethal host-vector system without antibiotics resistance gene screening .The ORF sequence of N gene was cloned by PCR amplification using specific primers designed according to the sequence of IBV SAIBk strain (serial number: DQ288927)and ligated to vector pYA3342 to construct recombinant plasmids pYA3342-N.Then the positive recombinant plasmid identified by PCR amplification and endonuclease digestion was introduced into S.typhimurium X4550 strain by the way of CaCl2.We constructed recombined attenuated Salmonella typhimurium X4550(PYA3342-GFP) which contained reported gene of green fluorescence GFP .Strains were oral immunization. We make frozen section of chicken organic tissue to observe the regular distribution of recombined strains by fluorescence microscope.The result showed that recombined strains can distribute and set in intestine, liver, spleen, kidney, lung and heart .It is the advanced and effective method used to inspect regular distribution of attenuated Salmonella typhimurium in chicken vivo.There is no adverse reaction and pathological change after feeding chinken by 1×10⁹CFU,5×10⁹,CFU1×10¹⁰CFU of different dosage, that testified the vaccine X4550(PYA3342-N) is safe. 6 weeks post-priming ,N gene in lung and kindey of chickens could be amplified by PCR of positiv colonies to evaluate stabililty.The results indicated that the IBV specitific antibodays were induced at 14day after immunizaion and was significant higher than PVAX1-N (P＜0.05). The mucoantibody IgA of intestinal tract can be detected after boosting three weeks and was significant higher than PVAXl-N (P＜0.01).The percentage of CD4⁺ and CD8⁺ T lymphocyte of the vaccine were significant higher than PVAX1-N and inactivated vaccine (P＜0.05). Furthermore ,the protection rate of X4550(PYA3342-N) group, inactivated vaccine group and PVAX1-N group was 73%, 80% and 67% , respectively. This study should encourage further work towards the development of recombined attenuated Salmonella typhimurium vaccine against IBV.