| Infectious bursal disease is a highly contagious,acute infectious disease caused by IBDV,mainly infecting 3～12-week-old chickens and turkeys,attacking the unique immune organ of birds,bursal sac,and infecting B lymphocytes in bursal follicles.Due to the severe immunosuppression caused by the decrease of B lymphocytes,the immune failure of a variety of vaccines and secondary infection have increased,which has increased the difficulty of disease prevention and control and caused huge losses to the poultry industry.In recent years,new variants have emerged,and the poultry industry is facing great challenges.Although different vaccines and methods to control the invasion of very virulent strains have been practiced,bursal disease still occurs from time to time,and the exploration and development of IBDV vaccines is still very necessary.Turkey herpesvirus HVT is a highly efficient recombinant vaccine vector that can express the main foreign genes of a variety of poultry diseases to prevent and control corresponding epidemics,and has been widely used for half a century.In this study,a recombinant HVT virus that can express VP2 protein was constructed by using CRISPR/Cas9 technology and nonhomologous recombinant(NHEJ)method.The biological characteristics of the recombinant virus and its immunoprotective effect were evaluated.1.Construction of recombinant turkey herpesvirus expressing IBDV infectious bursal virus VP2 protein at US2 site and with mCMV promoterVP2 gene was amplified by PCR from the very virulent IBDV Jiangsu strain(hereinafter referred to as JS strain)isolated in our laboratory,and linked to the mCMV promote.Then VP2 and eGFP gene expression cassette was respectively inserted into the non-essential region of the HVT genome at US2 site by CRISPR/Cas9 technology.The recombinant fluorescent virus was obtained after several rounds of screening and purification.eGFP tag was knocked out by the Cre/LoxP recombinase system.A recombinant HVT virus expressing IBDV’s infectious bursal virus VP2 protein was constructed and named rHVT-US2-mCMV-VP2.PCR identification confirmed insertion of the VP2 gene at the US2 site.The VP2 protein was expressed in IFA and Western-blot.The results of the growth curve of recombinant virus on CEF showed no different from that wild-type parental virus.The results of passage stability test of recombinant virus showed that the VP2 gene could still be stably expressed after 15 continueing passages of CEF.2.Determination of the immune efficience of recombinant turkey herpesvirus expressing IBDV infectious bursal virus VP2In order to investigate the immune efficience of the constructed VP2 recombinant turkey herpesvirus,1-day-old SPF chickens was immunized with recombinant virus.At 14 days old,the chickens were challenged with IBDV very virulent JS strain.The immune efficience was compared with the commercial vaccine Vaxxitek HVT-IBD.The results showed that rHVTUS2-mCMV-VP2 recombinant virus and commercial vaccine had good immune protection against IBDV very virulent JS strain.The protective effects were 100%.The bursa micropathological results showed that the protection rate of rHVT-US2-mCMV-VP2 was 75%and commercial vaccine Vaxxitek HVT-IBD was 84.6%. |
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