Construction And Characteristics Of Recombinant Attenuated Salmonella Typhimurium Expressing HN Of NDV And VP2 Of IBDV Fusion Protein

Newcastle disease(ND),the OIE list A disease,and infectious bursal disease(IBD),list B infectious diseases,caused significant economic losses to poultry industry worldwide.In China,ND and IBD have been a serious damage to the industry development.Newcastle disease,infectious strong,mortality is high,and the infected host range is widely.IBD mainly destory the humoral immune central organs-bursa of fabricius,resulting in impaired lymphocyte function,causing the immunosuppression of chicken,thus affecting the immune effect of Newcastle disease,avian influenza vaccine.It is imperative to develop a new type of vaccines for the prevention and control of ND and IBD due to appearring of NDV and IBDV virulent strains and variants currently.Attenuated salmonella as vaccine carriers is natural mucosal immunity boosters.It can present a variety of antigens and induce specific mucosal,cell and body fluid immunity response.In this study,we adopted asd+ host-carrier balanced lethal system,HN and VP2 genes were fused by SOEing PCR,and it was inserted into prokaryotic expression vector p YA3493.Then it was introduced into attenuated Salmonella typhimurium.The expression of HN and VP2 fusion protein recombinant attenuated salmonella vaccine candidates was successfully constructed,and its biological and immunobiological characteristics were studied preliminarily.It will lay a foundation for developing a novel vaccine of NDV-IBDV-S.typhimurium.1.Prokaryotic expression of fusion protein of NDV HN and IBDV VP2.The p MD18-T-HN and p MD18-T-VP2 were used as the template to amplify the HN gene fragment and the VP2 main antigen epitope region.The HN-VP2 fusion fragments were amplified by overlapping PCR SOEing and cloned into p MD-19 T,and the recombinant plasmid p MD19-T-HN-VP2 was constructed.After identification by PCR,double digestion and sequencing,the fusion gene fragment was ligated to expression vector p ET32 a,and was transferred to Rosetta strains of E.coli to express.The SDS-PAGE and Western blotting results showed that Fusion protein was expressed in Rosetta strains,the size is 104 k Da,and had good reactionogenicity.2.Construction and biological characteristic study of recombinant attenuated Salmonella typhimurium expressing HN of NDVand VP2 of IBDV fusion protein.The fusion gene fragments HN-VP2 were inserted into prokaryotic expression vector p YA3493.After identification by PCR,double digestion and sequencing,the recombinant vector p YA-HN-VP2 was introduced into attenuated Salmonella typhimurium three deletion strain SL1344?crp?cya?asd to obtain the attenuated recombinant bacteria SL1344?crp?cya?asd(p YA-HN-VP2),and its biological characteristics were studied preliminarily.Western blotting analysis demonstrated the HN-VP2 fusion protein was secretion expressed in attenuated recombinant bacteria,and the fusion protein can react with HN antibody,VP2 antibody respectively,and had good immunoreactivity.Biological characteristics results showed that the capabilities of the recombinant strains to use carbon sources were decreased,the growth rate became slowe,the LD50 was reduced at least 105 times compared with SL1344,and it had good invasiveness;recombinant plasmid p YA-HN-VP2 can be inherited stably.3.Immunological characteristics of attenuated recombinant bacteria SL1344?crp?cya?asd(p YA-HN-VP2)In order to determined the immunological response in chicken after recombinant bacteria SL1344?crp?cya?asd(p YA-HN-VP2)oral vaccination.The recombinant bacteria SL1344?crp?cya?asd(p YA-HN-VP2)was administered orally two times to chicken,each time interval of 7 d.And set the SL1344?crp?cya?asd(p YA-HN),SL1344?crp?cya?asd(p YA-VP2),empty carrie,PBS and NDV vaccine+IBDV vaccine as the control group.The growth of chickens,NDV antibodies,VP2 antibodies,intestinal mucosa s Ig A and lymphocyte differentiation level were detected after immunization of 7 d,14 d,21 d,28 d,35 d,42 d.And the protection of recombinant strains were evaluated.The results showed that: the recombinant had significant positive effects on the growth of immunity organs,there was no significant difference on the growth of chickens compared with control group(P>0.05);After immunization of 7 d,chickens in recombinant group began to produce VP2 antibodies and NDV HI antibody,and reached the highest in 28 d,which were obviously higher than control group(P<0.01);In s Ig A test,High level secretory Ig A was detected in intestinal rinses of recombinant group,and got the maximum at 28 day after immunization;In lymphocyte proliferation test,recombinant group can proliferate peripheral blood lymphocyte;After immunization at 7-42 d,the concentration of IFN-? higher than IL-4,so the immune response induced by recombinant bacteria tend to Th1 immune response.In the immunoprotection test,recombinant inmmunized chichens had66.67% resistance infected by NDV,77.33% resistance infected IBDV and 100%resistance infected by Salmonella typhimurium.In conclusion,the recombinant strain can induce effective humoral,muscosal and cellular immune responses after oral immunization.It provides a new method to the Vaccine development of ND and IBD,and it lays foundation for the prevention and control of ND and IBD.