Prokaryotic Expression Of Infectious Bursal Disease Virus VP2 Gene And The Study Of Its Anti-genicity

Infectious bursal disease(IBD), which usually occurs in 3-12 weeks old chicken and young chicken, is a highly contagious and severe immunosuppression disease caused by infectious bursal disease virus(IBDV). The virus destroys the bursa of Fabricius, leading to serious damage to other immune organs and cells as well as considerable economic losses in poultry industries. IBDV can encode five kinds of proteins, which are VP1, VP2, VP3, VP4 and VP5 respectively. VP2 is the main structural protein. It is on the outside of nucleocapsid and carries the mainneutrality epitope, which can induce host to generate neutrality antibody that will protect chicken from infecting by IBDV.In recent years, the prevention of infectious bursal disease face tremendous pressure due to the emergence of super strains and variant strains. So we need to develop new vaccine and made them become more safer and effective.The thesis ineludes 5 parts:First, bursals were collected from chickens that suspected suffered IBD in liaoning province areas where IBD broke out were grinded thoroughly, frozen and melted for several times, then centrifuged and the supernatant was collected. Virus was isolated after inoculated in chicken embryos then was subjected to agar diffusion test and Animal regression test, and one virus strain was identified.Second, SPF embryonating eggs were inoculated by the diluent, total RNA of virus was extracted from allantoic fluid by the method of Trizol reagent, according to the VP2 gene sequence accessed in Gen Bank, a Pair of Primers were designed. IBDV VP2 gene was amplified by RT-PCR, then cloned and sequenced. Sequence analysis showed that the homology with the strains publish in Genebank are 99% respectively, all these indicated that the field strain is vIBDV.Third, the target gene cloned into expression vector pET-28a, and the recombinant plasmids pET-VP2 was obtained. The insert position, the orientation and the ORF were verified by PCR, digestion and sequence analysis. Comparing with the published sequence of gene IBDV showed that they were homologous. The recombinant plasmids were transferred into BL21(DE3). The target protein was induced by IPTG. The results of SDS-PAGE and western blot indicated that the molecular weight of VP2 Protein was about 48 ku, and it had strong immunological activity.Fourth, fusion protein with the white oil adjuvant emulsion in accordance with the appropriate ratio, In order to made genetic engineering subunit vaccine.Fifth, according to the analysis of the protective efficacy of genetic engineering subunit vaccine by challenge protection experiment, The results showed that the protected rate was 80%.