Construction And Characteristic Study Of Recombinant Newcastle Disease Virus Haemagglutinin-neuraminidase Gene Attenuated Salmonella Enteria Serovar Pullorum

Newcastle disease(ND) is one of acute contagious avian disease caused by Newcastle disease virus(NDV), which has devastating destruction and was listed as one of notifiable terrestrial animal disease by the World Organisation for Animal Health. It is imperative to develop a new type of genetically engineered vaccines for the prevention and control of ND due to many defects with traditional vaccine for ND prevention recently. Salmonella enteria serovar Pullorum(S. Pullorum) is currently one of major bacteria which have great damage to the poultry industry. Drug control could easily lead to biosafety issuses, and immune protective efficacy of traditional vaccines was not high. Therefore, it could be a good choice to use attenuated Salmonella as novel live vaccine to control bacterial desease nowadays. The study was to explore the immunobiological characteristics of recombinant strain including a major protective HN gene of NDV which was carried by asd mutant balanced lethal system on the basic of C79-13Δcrp strain.1. Construction and biological characteristics of attenuated S. Pullorum mutant C79-13?crp?asdIn this study, the recipient strain S. Pullorum mutant C79-13?crp was transconjugated with the donor strain E.coli χ7213 contained p REΔasd. “Two-step method” was used to screen asd gene deletion strain. The results of PCR and sequencing showed that the S. Pullorum mutant C79-13?crpΔasd was constructed scuccesfully. The results of biological characteristics indicated that the constructed mutant loss the ability of living in DAP negative environment; The capabilities to use these carbon sources like maltose, glucose, manntiol, rhamnose were lost at the same time; Comepared with wild-type strain C79-13, the serotype of mutant C79-13?crpΔasd had no change, and growth rate of the mutant became slower; The mutant can carry Δasd fragment stably in vitro. The results of oral challenge in chichens manifested that the LD50 of C79-13?crpΔasd was increased about 3.1×104 times compared with C79-13.2. Construction and biological characteristics of recombinant NDV HN gene attenuated S. Pullorum mutantIn this study, HN gene fragment ampified by PCR was cloned into selectable marker-free prokaryotic expression vector p YA3493. Then we got recombinantexpression vector p YA-HN after identification of PCR and restriction enzyme digestion was transferred into C79-13?crpΔasd to construct recombinant C79-13Δcrp?asd(p YA-HN). Biochemical properties and growth characteristics of the recombinant strain are basically consistent with C79-13ΔcrpΔasd; NDV HN gene can be stably passage in vitro; The SDS-PAGE results showed that HN gene was expressed in recombinant strain, and Western blot analysis demonstrated the expressed protein had good immunogenicity; The virulence of the recombinant was was increased about 3.4×104 times compared with C79-13.3. Preliminary study of immunology characteristics of recombinant C79-13 Δcrp?asd(p YA-HN) strainThe immunology characteristics of recombinant strain had been preliminary studied through chichens’ immunization. These results proved that the recombinant strain can survive stabily in chichens which was highly invasive; There was no obvious difference in chickens’ weight increasing(P>0.05), significant difference in stimulating index of immunity organs(P<0.05); In HI antibody test, HI antibody level got the maximum(log2=5.3); Similarly, the Salmonella Ig G antibody level of recombinant group was obviously higher than control group(P<0.05) at 21 day after chichens’ s immunization; High level secretory Ig A(log2=3.56) was detected in intestinal rinses of recombinant group compared with PBS control group; The SI(stimulation index) of isolated lymphocytes from recombinant inmmunized chichens after stimulation by NDV in vitro got the maximum(SI=1.698) which difference was siginificant(P<0.05) compared with PBS control group; In the immunoprotection test, recombinant inmmunized chichens had 100 % resistance infected by wild-type strain and 60 % resistance infected by NDV F48E8. In conclusion, the recombinant strain can stimulate organism to produce effective humoral immune responses, muscosal immune responses and cellular immune responses at certain time after immunization, and it had a protective efficacy against infection of wild strains of S. Pullorum and NDV.