| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === The objective of the present study was to investigate the feasibility of rVP2H proteins produced by insect larvae to protect chickens against infectious bursal disease virus infection. Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease in young chickens.
The recombinant protein rVP2H has an extra six-histidine residue at the C- terminus of the VP2, a structural protein of IBDV and the primary host-immunogen containing the antigenic regions responsible for elicitation for neutralizing antibodies. The rVP2H self-assemble to form virus—like particles (VLPs) when expressed in Trichoplusia ni larvae which could be purified by either ammonium sulfate precipitation or IMAC (immobilized metal-ion affinity chromatography).
The protective efficacy of the purified rVP2H particles was evaluated using a chicken protection assay. VLPs administered intramuscularly with CFA to SPF (specific-pathogen-free) chickens induced higher neutralizing antibodies than commercial vaccine. At dpi 28, injecting VLP vaccine about 4-folds high than the VN titer induced by injecting commercial vaccine. All chickens injecting VLPs were protected from challenge; no virus in bursal was shed by AGP and Western blotting after challenge. Otherwise, the dosage of 20 μg、2 μg、0.2 μg rVP2H injected to SPF chickens provide the protection against the 10000-folds of LD50 IBDV challenge. No virus in bursal was detected by AGP and Western blotting, too.
The 2 μg rVP2H product from larvae treatment by 50 % ammonium sulfate precipitation absence IMAC purification be the subunit vaccine test. The SPF chickens injected the kinds of subunit vaccine induced the protection against 10000-folds of LD50 very virulent IBDV challenge. No virus in bursal was shed by AGP and Western blotting. Additionally, the recombinant protein rVP2H administered orally induced protection from LD50 very virulent IBDV challenge in 25 ~ 75 % in chickens.
Under the condition test, the low dosage of 0.2 μg purified rVP2H are immunogenic when administered intramuscularly to SPF chicken addition of CFA. The efficacy dosage of cheaper subunit vaccine prepared by ammonium sulfate precipitation is 2 μg.
In conclusion, the expression level of rVP2H is 5 mg per Trichoplusia ni. Following ammonium sulfate precipitation, approximately 4 mg / larvae was obtained. Thus, the amount of rVP2H (4mg) produced by one larvae was able to immunize 2000 chickens and protected them from infection of a strain of very virulent IBDV. On other hand, 800μg / larvae of rVP2H protein was obtained following the purification using IMAC, which was able to immunize 4000 chickens and protect them from infection of a strain of very virulent IBDV.
|
|---|
