| Bovine infectious rhinotracheitis is an acute infectious disease caused by infectious bovine rhinotracheitis virus.Severe respiratory diseases,miscarriage and other neurological diseases are commonly seen in cattle.It is difficult to prevent and clear the disease due to latent infection created by IBRV.Currently,vaccination is still an important strategy to prevent and control the disease associated with IBRV.IBRV vaccines mainly include inactivated vaccines,attenuated vaccines and genetically engineered vaccines,but all represent various defects.Therefore,it is necessary to develope a more efficacious vaccine candidate,especially for pregnancy cow feedlots.Compared with the traditional genetic engineering subunit vaccines,multi-epitope vaccine enables to overcome the limitations of MHC class molecules and make it efficiently presented to lymphocytes,and can effectively cope with the variation of pathogenic microorganisms.Therefore,the IBRV multi-epitope chimeric protein was expressed and immunogenicity was evaluated in this study in an attempt to generation of IBR vaccine candidate,which might be potential of use in pregnant cows and heifers.IBRV g B,g C,g D epitopes and universal toxin T-cell epitope P2 were linked with GGGGS or AAYAAY,and the antigenicity of recombinant chimeric protein was analyzed by DNASTAR Protean software.The best combination of gene fragments was synthesized,and was cloned into expression vector pET-28a(+).The recombinant plasmid was transformed to E.coli BL21(DE3)competent cells,and the recombinant proteins were expressed and identified by SDS-PAGE and Western blot.The expressed chimeric protein was purified by High Affinity Ni+-NTA Purification System and emulsified with ISA206 to vaccinate Chinese white rabbits three times at 3-week intervals.The level of sera raised by recombinant proteins was determined on 21,42,63 days post immunization.The result showed that the chimeric protein was expressed successfully in the form of inclusion body,and antibody levels induced by chimeric protein linked by AAYAAY was higher than that by chimeric protein linked with GGGGS.Based on the P2-g B/g C/g D sequence linked with AAYAAY,pET-28a-P2-g B/g C/g D-Bo IL-6,pET-28a-P2-g D-Bo IL-6 and pET-28a-Bo IL-6 recombinant plasmids were constructed.Meanwhile,RNA was extracted from the spleen of Chinese white rabbits,and the rabbit IL-6 gene was amplified by RT-PCR.The amplified rabbit IL-6 was cloned into pET-28a-P2-g B/g C/g D to construct a pET-28a-P2-g B/g C/g D-Ra IL-6 recombinant plasmid.The positive recombinant plasmid was transformed into E.coli BL21(DE3)competent cells to induce the expression of the recombinant protein.The recombinant protein was purified and analyzed by SDS-PAGE and Western blot.The results showed that pET-28a-P2-g B/g C/g D-Bo IL-6,pET-28a-P2-g D-Bo IL-6,pET-28a-Bo IL-6 and pET-28a-P2-g B/g C /g D-RIL-6 plasmid were expressed in the form of inclusion bodies,and the purified recombinant proteins reacted specifically with anti-His-Tag monoclonal antibody.The each of purified recombinant protein was emulsified with ISA206 adjuvant to immunize the Chinese white rabbits,and the IBRV-BVDV inactivated vaccine group and the ISA206 adjuvant control group were set as the controls.Immunization was performed three times at 3-week intervals.The level of antibody and cytokines IL-4 and IFN-? were determined using indirect ELISA.Three weeks after third immunization,rabbits are challenged by IBRV.The nasal swab was collected and the body temperature of immunized rabbits was measured at 1st,3rd,5th and 7th day post-challenge of IBRV.The real-time quantitative PCR was established to detect the copies of IBRV in the nasal swab and lung.After 7 days,one rabbit was euthanized,and lung and trachea were taken to make pathological tissue sections.Dexamethasone(0.1 mg/kg)was injected intramuscularly into the rabbits in each groups for 5 days to activate putatively latent virus at 30 days post-challenge of IBRV.Nasal swabs were collected from the rabbits on days 1st,3rd,5th and 7th after injection of Dexamethasone to detect viral shedding.After 7 days,the rabbits were euthanized and,lung and trachea were collected to prepare histopathological section.The results showed that the antibody levels induced by P2-g B/g C/g D-Bo IL-6 were significantly higher than other recombinant proteins at 21 d,42 d and 63 d(P <0.05),Which was lower than the IBRV-BVDV inactivated vaccine group(P <0.001).In addition,the neutralizing antibody titer induced by P2-g B/g C/g D-Bo IL-6 were significantly higher than other recombinant proteins 42 d and 63 d(P <0.05),and there was no significant difference with the IBRV-BVDV inactivated vaccine group.Cytokine assay showed that P2-g B/g C/g D-Bo IL-6 can induce IL-4 and IFN-? effectively compared with the other groups,which indicated that Th1 and Th2 immune responses was induced by P2-g B/g C/g D-Bo IL-6.Virus copies in the nasal swabs were measured,which showed that the virus was detected in the nasal swab at 1st,3rd and 5th day post challenge by IBRV.There was no significant difference between P2-g B/g C/g D-Ra IL-6 and g B/g C/g D-Bo IL-6 group at 1st day,while difference was significant on the 3rd and 5th day.The difference between g B/g C/g D-Bo IL-6 and other recombinant proteins was significant(P <0.05),and there was no significant difference with the IBRV-BVDV inactivated vaccine group.In addition,virus copies in the lung of P2-g B/g C/g D-Bo IL-6 group was significantly lower than other recombinant proteins 7 days post-challenge,(P <0.05),and there was no significant difference with IBRV-BVDV vaccine group.Pathological section showed that there was a little amount of inflammatory infiltration in the lungs from P2-g B/g C/g D-Bo IL-6 group,and there was no pathological changes in the trachea.Obvious damage was not observed in the lung and trachea from IBRV-BVDV inactivated vaccine group.After reactivation induced by dexamethasone,there was no significant difference on body temperature between the experimental groups.Virus copies in the nasal swabs from P2-g B/g C/g D-Bo IL-6 group was significantly lower than other recombinant protein groups.However,there was no significant difference with the P2-g B/g C/g D-Ra IL-6 group and the IBRV-BVDV inactivated vaccine group on 1st,5th day.Virus copies of lungs was significantly lower than other recombinant protein groups(P <0.05).Pathological section showed that there was a amount of inflammatory infiltration in the lungs from P2-g B/g C/g D-Bo IL-6 group,and no obvious damage were found in the trachea.Lymphatic hyperplasia was observed in the lungs of IBRV-BVDV vaccine group,and there were no changes in the trache.The study showed that the rigid AAYAAY linker was superior to GGGGS for the generation of IBRV multi-epitope recombinant chimeric protein.Furthermore,Bo IL-6 can effectively improve the immunogenicity of multi-epitope chimeric protein,and improve protection against IBRV,which will be potential of the IBRV genetic-engineering subunit vaccine candidate. |
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