| Summary: | 碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 98 === Infectious bursal disease virus (IBDV) and Infectious Bronchitis virus (IBV) causes acute and contagious diseases in chickens with significant impact on the poultry industry worldwide. IBDV is a non-enveloped virus with icosahedral symmetry that contains two segments of double-stranded RNA. The large segment encodes for a polyprotein, VP243, further processing to VP2, VP4 and VP3 protein. VP2, a structural protein of IBDV, can induce neutralization antibody against IBDV. IBV contains a 27.6 kb single-stranded, positive-sense RNA genome and it encodes four major structural proteins. S protein is synthesized as a large protein that is post-translationally processed to the S1 and S2 subunits. The primary neutralization epitopes of S are found on the outer globular-like S1. The purpose of this study is to develop a divalent vaccine against both of IBDV and IBV. In trial one, the VP2 gene and/or S1 gene were cloned into pcDNA3.1 or pET28b vector for protein expression as DNA vaccine or subunit vaccine, respectively. S1 gene was cloned to the N-terminal of VP2 to produce fusion proteins of S1-VP2. One-day-old specific-pathogen-free chickens were vaccinated with 500 μg of prepared DNA vaccines three times at weekly intervals or with 200 μg of proteins two times at bi-weekly intervals. Chickens were challenged with IBDV or IBV at four weeks of age. In trial one, for IBV study, chickens received vaccines showed positive but low anti-IBV ELISA titers before challenge. After challenge, chickens received S1-VP2 fusion protein had 20 % mortality and chickens in challenge control had 60% mortality. For IBDV study, the ELISA titers of vaccinated groups are also positive with titers as high as 4600. After challenge, chickens received VP2 or S1-VP2 DNA had bursal lesion score 2.4 and 2.6, respectively, and chickens in control groups had lesion score 4. The results of trial 1 showed that both DNA and subunit protein vaccines expressing S1, VP2 or S1-VP2 proteins were able to induce specific antibody to related antigens. However, the protection efficacies of constructed vaccines against IBD and / or IB were low. In order to improve the efficacy of vaccines, pTriEx vector was used instead of pcDNA3.1 or pET28b and S1 gene was fused to VP2 or VP243 on the C terminal instead of N terminal. Trial 2 of animal study was conducted. For IBV study, no matter of chickens received vaccines or not, chickens showed no anti-IBV ELISA titers before challenge. After challenge, chickens received VP2-S1 or VP243-S1 fusion protein had 17 % and 0% mortality and chickens in challenge control had 33 % mortality. For IBDV study, chickens received DNA vaccines of VP2, VP243 or VP243-S1 showed significant higher anti-IBDV ELISA titers when compare to those in NC group. After challenge, chickens received VP243 or VP243-S1 DNA had bursal lesion score 1.17 and 2.5, respectively, and chickens in control groups had lesion score 4. The result indicated that both DNA vaccines pTriEx-3/VP243 and pTriEx-3/VP243/S1 were able to induce protective humoral immune responses in chicken against IBDV challenge. However, the constructs of IBV-S1 gene alone or fusing to VP2、VP243 gene did not induce ELISA antibody titers and not provide any protection against IBV challenge.
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