Immunogenicity Of Recombinant Protein Co-expressed By MChIL-18 Gene With VP2 Gene Or HA1 Gene In PFastBac^TM Dual

Interleukin-18(IL-18) is a novel pleiotropic cytokine, which has the potent inducor of IFN-γproduction.It has been demonstrated that IL-18 plays a major role in immunoregulation and protection, and has large potential in host defense against various micro- organic infection and tumor. So IL-18 which was as a kind of efficient immune adjuvant for vaccine, can coexpress with some protective gene of pathogenic microorganism, thereby enhance and improve the immune effective of vaccine. However, the study on chicken interleukin-18 (ChIL-18) lagged off the mammal interleukin-18. Previously, the gene of ChIL-18 was cloned, but further study is not performed.The discovery of ChIL-18 gene is very meaningful for both the study on comparative immuneology and the therapeutics for poultry diseases. pFastBac^TM Dual has the able to express simultaneously two active foreign proteins, because it has PH promoterand p10 promoter. The purification of recombination protein is possible because of inlet of His tag.Infectious bursal disease (IBD) causes considerable economic losses to the poultry industry worldwide by causing a high rate of morbidity and mortality in an acute form or as a consequence of severe immunosupression provoked by the destruction of immature B-lymphocytes within the bursa of Fabricius. Vaccination against the disease with inactivated or attenuated live vaccines is widely practiced. However, such traditional vaccines have become less effective due to the emergence of very virulent or antigenic variant strains of IBDV in recent years.VP2 and VP3 are major capsid proteins, whereas VP2 is the major antigen of IBDV and contains major epitopes responsible for protection against IBDV. VP2 can stimulate neutralizing antibodies and accordingly induce humoral immuniny in vivo. Therefore, the VP2 protein has been used quite effectively in developing subunit/DNA vaccines with other expression system against IBDV. Protein immunization opens a new approach to the development of gene vaccines for chickens against infectious diseases.Some of the most highly contagious viruses of human and veterinary concern are airborne pathogens, e.g. human and avian influenza virus (AIV). The highly pathogenic avian influenza viruses (HPAI) have been a threat to the poultry industry for many years. However, when the Asian H5N1 subtype emerged, a trans-boundary spread became evident and a new dimension of zoonotic danger and fear for evolution of a pandemic virus was raised. However, traditional vaccines have become less effective due to the emergence of very virulent or antigenic variant strains of AIV years. So a newly vaccine and its adjuvant are urgently demand to research. And major antigen determinants of AIV lie in HA1 gene, so HA1 protein is the ideal electing antigen of studying gene engineering subunit vaccine.In the experiment, the mature chicken interleukin-18(mChIL-18) gene and VP2 gene/HA1 gene were amplified on the basis of pMD18-T-VP2 plasmid,pMD18-T-HA1 plasmid,pMD18-T-mChIL18 plasmid. Then mChIL-18 gene and VP2 gene/ HA1 gene were inserted into the downstreams of PH promoter and P10 promoter of baculovirus expression plasmid pFastBacTM Dual, respectively. Finally, the constructing eukaryotic expression plasmid pF-IL18,pF-VP2,pF-IL18-VP2,pF-HA1,pF-IL18-HA1 were transformed into DH10Bac competent cells, and screened by three antibiotics (kanamycin, gentamycin and tetracycline) and blue-white patch. The recombinant bacmid DNA is extracted, on the basis of M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis), we analyze rBac-IL18,rBac-VP2,rBac-IL18-VP2,rBac-HA1,rBac-IL18-HA1 DNA by PCR.In order to harvest the P1 recombinant aculovirus, we transfected the purified bacmid into Spodoptera frugiperda 9(sf9) by using a cationic lipid such as Cellfectin Reagent. Raised the virus titre by infecting sf9 many times and infected the sf9 with the higher baculoviral stock (10⁷¹0⁸ pfu/mL) for expressing protein and harvested the supernatant and cells in different times. The expressed recombinant protein was analyzed by SDS– PAGE and IFA. The results demonstrated that IL18 protein and VP2 protein/HA1 protein were simultaneously expressed in sf9 cells.The biological activities of pIL18,pVP2-IL18 and pHA1-IL18 were detected by the proliferation of lymphocyte, inducing IFN-γproduction and inhibiting the Vesicular stomatitis virus (VSV). The experiment of MTT showed that every concentration of pIL18,pVP2-IL18 and pHA1-IL18 could enhance proliferation of lymphocyte, and the effect was best when the concentration was 200ng/mL and the proliferation exponent was 4.13,4.35,4.09, respectively. The experiment of VSV inhibition indicated that pIL18,pVP2-IL18 and pHA1-IL18 could induce spleenocytes to produce IFN-γwith high activity when it was more than 100ng/mL, and the amount of recombinant proteins and their inducing effect on IFN-γhad a direct proportion. In the experiment of VSV inhibition, different dilution of IFN-γwas used and they could protect the cells when the concentration was more than 10U/mL. The result showed that pIL18,pVP2-IL18,pHA1-IL18 could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. So it can be concluded that pIL18,pVP2-IL18 and pHA1-IL18 emerge functional activities by IFN-γ.Two-week-old SPF chickens were randomly divided into 7 groups with 20 chickens per group. Group I received B78. Group II received pIL18 and B78. Group III received pIL18 and pVP2. Group IV received of pVP2-IL18. Group V received B78 in primary immunization and pVP2-IL18 in booster immunization. Group VI received pVP2. Group VII served as PBS control and the normal control. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement. The level of cellular immunity was detected by FACS. The number of CD⁴⁺ and CD⁸⁺ T lympholeukocytes in peripheral blood of chickens immunized with genetically engineered vaccine were obviously higher than the control group and only B78 vaccine immunity class, which demonstrated satisfactory cell-mediated immunity (CMI) induced by genetically engineering vaccine. Group IV was always higher than group III and group I, and then presented significant difference. The number of CD⁴⁺ T lympholeukocytes in pVP2-IL immunized group kept increasing tendency and longer persistence comparing with the B78 group after the secondary immunization. The level of humoral immunity was detected by Infectious Bursal Disease Virus Antibody Test Kit. The anti-IBDV antibody titers of all experiment groups were presented upgrade tendency after secondary immunity and up to maximum in 4 weeks post inoculation. The antibody against IBDV detected in six chickens of group IV was always higher than other groups at days 7, 14, 21, 28 and 35 post-immunization. It presented significant difference (P<0.05) between group IV and group I at days 14, 21, 28 and 35 post-immunization. In addition, it also presented significant difference (P<0.05) between group IV and group III at days 7, 14, 21, 28 and 35 post-immunization.One-week-old SPF chickens were randomly immuned with pIL18,pHA1 and pHA1-IL18. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement. The results showed that compared with pre-immunization, all experiment groups promoted the proliferation of CD⁴⁺ and CD⁸⁺ T lympholeukocytes. The results showed that compared with pre-immunization, all experiment groups had higher titers of antibodies and maintained high titer levels for longer times.