Study Of Genetic Engineering Infectious Bursal Disease Vaccine And Two Immunopotentiators

Infectious bursal disease(IBD)is an acute,highly infectious lymphoid disease of young,sexually immature chickens,which is distributed worldwide.The disease caused ’by infectious bursal disease virus(IBDV),is characterized by destruction of lymphocytes in the bursa of Fabricius(BF).The outbreaks of IBD cause not only serious direct economic loss from high mortality but also large indirect economic loss from the decrease of animal laying performance and meat performance.Vaccination against IBD with inactivated or activated live viruses play an important role in preventing the disease,however,there are some defects of these vaccines,such as repetitive injection,viral pollution of the environment during vaccine production and immunization process.In addition,traditional vaccines are not totally effective against very virulent strain of IBDV(vvIBDV).Therefore,we adopt the following strategies to decrease the incidence of IBD in chickens:(1)Development of genetic engineering IBD vaccines y;(2)Study of recombinant hybrid bursin peptides;(3)Exploring of recombinant chicken interferon beta.1.Development of genetic engineering IBD vaccinesAlthough traditional active or inactive vaccines play a key role in preventing IBD,there are some defects,such as repetitive injection,and viral environmental pollution of the environment during viral production and immunization.Exploring efficient vaccines using recombinant IBDV antigen and a subunit multi-epitope peptide may be a strategy to overcome these defects.However,protein antigen and multi-epitope peptides always have low immunogenicity,therefore we made the following attempts to improve immunogenicity:(1)Study of recombinant IBDV subunit antigen protein vaccine:VP2 antigen protein is a host protective antigen,which induces virus neutralizing antibodies that protect susceptible chickens from IBDV infection.To improve the VP2 immunogenicity,we cloned IBDV VP2 gene with one epitope peptide(KFDQML)codes at the N terminal and another epitope peptide(LASP)codes at the C terminal,which was named IBDV mVP2Owing to a successful expression of recombinant yeast Hepatitis B vaccine,the mVP2 gene was firstly expressed in yeast.Results showed that we have successfully expressed glycosylated IBDV mVP2 protein in the methylotrophic yeast Pichia pastori.However,the expression level of the mVP2 Protein was very low,which didn’t accord with the low cost meets of avian medicine.Then we selected the Escherichia coli expression system to express the mVP2 Protein,results suggested that the expression level of the mVP2 Protein is 20 times greater than that in yeast system.Agar-gel precipitation antigen titer of IBDV mVP2 expressed in Escherichia coli is up to 1:16,indicating the ideal immunoreactivity of IBDV mVP2.To further study the immunogenicity,ten-day-old SPF chickens were vaccinated with IBDV mVP2 subunit antigen protein vaccine.The results showed that the pathological lesions of bursa of Fabricius in vaccinated chickens are much better than that in positive control chicken.However,compared to the negative control,the protection of mVP2 vaccine was limited.All these results indicated that the mVP2 we produced was not an ideal antigen for vaccine.Although we have tried several ways to improve the mVP2 immunogenicity,such as using of Freund’s adjuvant white oil adjuvant together with the genetically engineered hybrid bursin and chicken interferon-β,the results didn’t show great improvement.As another point of view,we considered that the mVP2 protein had the potential to be IBD diagnosis antigen because of its high agar-gel precipitation titers.(2)Development a subunit multi-epitope peptide vaccine:In this study,three IBDV tandem multi-epitope peptide sequences were linked in series by SOE PCR and expressed in E.coli.The recombinant IBDV SOE multi-epitopes were highly expressed at yield about 18%of total protein in IPTG-inducible Escherichia coli BL-21(DE3)expression system.We hope that the recombinant multi-epitope peptide antigen could be a potential epitope-based vaccine for prevention of the IBD.Our results showed the high immunoreactivity(AGP antigen titer is 1:32),however,we are dissatisfied with the animal immune protective effect.2.Study of recombinant hybrid bursinBursin,a tripeptide(Lys-His-Gly-NH2)hormone,is well known to induce B cell differentiation.The 14 repeated bursin and 2 Gagnon’s peptide(Lys-Asn-Pro-Tyr,Stimulation of B-cells)tandem nucleotide sequence were synthesized and expressed in E.coli.The results showed that the expressed hybrid bursin was accumulated up to more than 20%of total bacterial protein.We then purified the recombinant hybrid bursin.Lymphocyte transformation test results showed that 0.9mg/ml purified recombinant enzyme-treated bursin(with elastase)can successfully induce chicken peripheral blood B lymphocyte cell proliferation.Results presented in animal experiment demonstrated that recombinant bursin could significantly increase 4-fold AGP antibody titers in the serum from the chickens vaccinated with inactivated IBD vaccine.Pathological analysis of bursa of Fabricius showed that genetic engineering bursin could improve the pathological lesions of BF.Recombinant bursin can also protect the chicken bursa of Fabricius against pathological lesions induced by IBDV.3.Study of recombinant chicken interferon betaInterferon beta(IFN-β)is a class of natural protein with a broad range of biological activities such as inhibition of virus replication.In this study,chicken interferon beta gene was cloned and expressed in E.coli.SDS-PAGE illuminated that the expressed protein is about 28 kD in size which accounedt for 18%of total protein of host cell.Cytopathic effect(CPE)inhibition assay showed that recombinant chicken interferon beta exhibited significant antiviral activity against IBDV in 24h and long-lasting activity against vesicular stomatitis virus(VSV).Animal experiment results showed that recombinant chicken interferon beta could enhance anti-IBDV activity of chicken vaccinated with inactivated IBD vaccine.In our work,recombinant IBDV protein vaccine and multi-epitope peptide have high immunoreactivity but low immunogenicity.These two subunit IBDV antigen protein and multi-epitope peptide have the potential applied value if there are used as standard AGP antigen in detecting IBD.Two recombinant immune enhancements can significantly improve the chicken immunity against IBDV and have promising applications.