| Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV), leading serious economic loss for poultry industry. Currently used live-attenuated ILTV vaccines could regain virulence after consecutive passages in chickens, leading the outbreak of ILT. It is necessary to establish a method for detecting antibody of ILTV, as well as development of a new generation of ILT vaccines safely and efficiently.In this research, we firstly amplified the gD gene fragment (253nt～1218nt) from the ILTV Anhui-2011-2genomic DNA based on prediction of signal peptides and analysis of transmembrane domains, then the fragment was cloned into pFastBacHTA plasmid and transform it into DH10Bac competent E.coli to generate a recombinant bacmid (Bacmid-gD). The Bacmid-gD was transfected into Sf9insect cell line by liposome-mediated method to generate a recombinant baculovirus AcMNPV-gD. Western-blotting analysis approved that AcMNPV-gD expressed a50KD recombinant protein. The recombinant gD protein was purified with High-Affinity Ni-NTA Resin, and then used as coating antigen to establish the indirect ELISA for the detection of ILTV antibody. The indirect ELISA showed high stability and no cross reaction with sera against NDV, IBV or AIV. Compared with BioChek ILTV ELISA Kit, the coincidence rate was93.02%.We evaluated the protection efficacy of recombinant gD protein by setting subunit vaccine group (Sv-gD), recombinant Marek’s disease virus vaccine group (rMDV-gD), combined immunization vaccine group (rMDV-gD+Sv-gD), live-attenuated vaccine group (K317) and non-vaccinated control group (Nv-ch, positive control). SPF chickens of recombinant Marek’s disease virus vaccine group and combined immunization vaccine group were immunized with5000PFU rMDV-gD per chicken in1-day-old, at21days of age, subunit vaccine group and combined immunization vaccines group were immunized with50μug subunit vaccine per chicken, while live-attenuated vaccine group immunized with one dose of K317. All the experimental groups were challenged with105EID50ILTV WangGang strain at the day of42. The morbidity and mortality of combined immunization vaccine group (27%and13%) was significantly differences (p<0.05) compared with the non-vaccinated control group (100%and67%), respectively. Significantly higher weight gain (p<0.05) after infection was observed in chickens of combined immunization vaccine group compared with live-attenuated vaccine group. Combined immunization vaccine group have significant differences (p<0.05) in clinical scores after infection compared with subunit vaccines group and recombinant Marek’s disease virus vaccines group. Overall, the protective efficacy of recombinant Marek’s disease virus for control of ILT could be enhanced by gD subunit vaccine. |
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