Construction And Characteristic Of Recombinant Attenuated Salmonella Enteria Serovar Pullorum Harbouring HN/F Genes Of Newcastle Disease Virus

Newcastle disease(ND),classified as Class A infectious disease by OIE,is an acute avian infectious disease.The use of traditional vaccines is greatly limited due to continuous emerging of NDV variants and atypical Newcastle disease.DNA vaccine is a novel ideal to develop ND vaccine.Fusion protein(F)gene and hemagglutinin-neuraminidase protein(HN)gene are the main protective antigens genes of NDV and become a new research hotspot for disease control and prevention.Salmonella enteria serovar pullorum(S.Pullorum)is still one of the most important pathogens and has great damage to the poultry industry.Traditional drug therapy easily leads to the emergence of bacterial resistance.The virulence of Salmonella attenuated by genetic engineering has declined evidently,but still has a good invasiveness and immunogenicity.The eukaryotic plasmid harbouring foreign genes can be carried into the animal cell by Salmonella natural infection,and can effectively produce mucosal immunity,cellular immunity,and humoral immune response.Attenuated Salmonella as a vaccine live carrier has a broad application prospects.In this study,a balanced lethal eukaryotic expression system of attenuated S.Pullorum strains carrying NDV HN/F genes was constructed,and further the biological and immunological characteristics of recombinant strains were investigated.1.Construction and characterization of a novel balanced lethal system based on attenuated Salmonella pullorumA new non-antibiotic eukaryotic recombinant plasmid renamed as pc D-asd was constructed by replacing the ampicillin resistance gene Amp with the asd gene of Salmonella in the pc DNA3.1-Zeo(+)plasmid.The complementary plasmid pc D-asd was electrotransformed into ?crp?asd C79-13 to construct the ?crp?asd C79-13(pc Dasd)balanced lethal system.The reporter gene-enhanced green fluorescent protein(egfp)gene was cloned into pc D-asd to construct the recombinant strain?crp?asd C79-13(pc D-asd-egfp).The system of biological characteristics and the ability of delivery foreign antigens were further investigated.Biological characteristics showed that the serotype of ?crp ?asd C79-13(pc D-asd)was identical to?crp?asd C79-13 and C79-13;the biochemical characteristics of ?crp?asd C79-13(pc D-asd)were basically the same with ?crp?asd C79-13 strain,but significantdifferent with the wild strain C79-13;the growth rate was also more slowly;the virulence of the recombinant stain was about 104 times lower than that of C79-13.Western blot analysis showed that the size of the foreign protein was about 27 KDa in CEF cells after infection with the recombinant strain.These results demonstrated that the new recombinant Salmonella pullorum ?crp?asd C79-13(pc D-asd)was constructed successfully.2.Construction and biological characteristics of recombinant attenuated Salmonella pullorum harbouring HN/F genes of Newcastle disease virusHN/F genes fragment ampified by RT-PCR were cloned into the CMV promoter downstream of the selectable marker-free eukaryotic expression plasmids pc D-asd.Then the recombinant expression plasmids pc D-asd-HN and pc D-asd-F(pc D-asdHN/F)identificated by PCR and restriction enzyme digestion were electrotransformed into ?crp?asd C79-13 to construct recombinant ?crp?asd C79-13(pc D-asd-HN/F).And the biological characteristics of recombinant strains and their ability to present HN / F protein were determined.Biochemical properties and growth characteristics of the recombinant strain were basically consistent with ?crp?asd C79-13;the recombinant strains were able to carry the HN / F gene stably in vivo and in vitro;the virulence of the recombinant stain was about 104 times lower than that of C79-13;Western blot analysis showed that the size of the HN / F protein were about 63 KDa and 60 KDa in CEF cells after infection with the recombinant strain.The results indicated that the recombinant NDV HN/F genes attenuated S.Pullorum mutant was successfully constructed,which can present and express NDV HN/F antigen genes in CEF cells.3.Immunology characteristics of recombinant attenuated Salmonella pullorum harbouring HN/F genes of Newcastle disease virusThe chickens were orally immunized with the recombinant strains to explore the immunological properties.These results proved that there was no obvious difference in weight increasing(P>0.05),significant difference in stimulating index of immunity organs(P<0.05);the level of NDV antibody was increased after 3 weeks,which significantly higher than the control group(P <0.05).The antibody level of the recombinant strain HN group was higher than the recombinant strain F group,but no significant difference;the level of Salmonella Ig G antibody was increased after 3weeks,which significantly higher than the control group(P <0.05);the level of s Ig Aantibody was increased after 3 weeks,which significantly higher than the control group(P <0.05).The antibody level of the recombinant strain HN group was higher than the recombinant strain F group,but no significant difference;The SI(stimulation index)of isolated lymphocytes increased slowly after immunization and reached the peak at 3 weeks,which significantly higher than the control group(P <0.05);the level of IFN-? was increased after 4 weeks,which significantly higher than control group(P<0.05).In the immunoprotection test,the protective rate of the recombinant strain HN/F group had 100% resistance infected by wild-type strain C79-13 and 70%,65%resistance infected by NDV F48E8.In conclusion,the recombinant strain?crp?asd C79-13(pc D-asd-HN/F)could produce effective humoral immune responses,muscosal immune responses and cellular immune responses after immunization,and have a considerable protection against infection.