雞傳染性貧血不活化病毒與重組蛋白之免疫原性分析

Immunogenicity analysis of an inactivated chicken infectious anemia virus and its recombinant proteins

黃暉傑(Huei-Jie Huang)

指導教授 ： 連一洋

國立屏東科技大學/農學院/獸醫學系所/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00283

摘要

雞傳染性貧血 (chicken infectious anemia; CIA)是由雞傳染性貧血病毒 (chicken infectious anemia virus; CAV)所造成，主要會侵犯幼齡雞隻造成貧血、免疫抑制、生長遲滯、產蛋下降等症狀，使得養雞業面臨重大的經濟損失。CIA雖有市售減毒活毒疫苗可供使用，但疫苗仍因毒力過強及具垂直傳染風險而無法施用於雛雞與產蛋中之種母雞身上。現有商用疫苗僅被建議在種雞8週齡至開產前4週間施打，而3週齡移行抗體消失時至10週齡主動免疫產生的間期，卻成為CAV野外強毒入侵的最佳機會。有鑑於此，本研究的目的在於研發一種不活化或次單位疫苗，藉以填補此段免疫的空窗期。試驗先進行CAV之VP1與VP2 基因選殖工作，將原已選殖在pGEX-4T-1載體但後續純化不理想之重組片段重新接合至表現載體pGEX-6P-1上，經由限制酶切割與電泳分析，確認其基因片段為預期之1368 bp VP1與651 bp VP2基因。轉型至C41 (DE3)原核表達系統進行重組蛋白表達，以抗GST 抗體以及CAV高力價血清進行西方墨點法，得到80 kDa VP1 與51 kDa VP2蛋白，證明其具有抗原性。在疫苗有效性試驗方面，評估單獨或合併去活化病毒與VP1與VP2表現蛋白免疫雞隻，動物實驗顯示免疫不活化混合表現蛋白組（iCAV+VP1/VP2）和 VP1/VP2組，於病毒攻擊後病徵輕微，且ELISA測定上較其他組別佳。顯示此種組合有較佳的免疫效力，頗有發展成為CAV次單位疫苗之潛力。

關鍵字

雞傳染性貧血病毒 ； 重組蛋白 ； 不活化病毒

並列摘要

Chicken infectious anemia (CIA), caused by chicken anemia virus (CAV), mainly resulted in anemia, growth retardation and immune suppression in young chicks. The disease causes a significant economic loss to the poultry industry. Current vaccine strategies are based on the prevention of vertical transmission in breeder and giving maternal antibody to chicks against wild CAV infection. Vaccination should be performed at about 8-15 weeks of age to avoid the risk of CAV vaccine strain spread through the egg. However, the age between 3 weeks and 10 weeks is weak protection against CAV due to degradation of maternal antibody and lower titer of activated immunity. The purpose of this study is to develop an inactivated vaccine or subunit vaccine for using in breeder and chick stages. This study re-cloned CAV VP1/VP2 gene to pGEX-6P-1 expression vector, through restriction enzyme digestion and gel electrophoresis to confirm the DNA fragments as 1385 bp (VP1) and 651 bp (VP2), respectively. Subunit protein production using the prokaryotic expression system that could be identified by Western blotting with anti-GST monoclonal antibody and CAV positive sera proved that antigenicity by the MW of protein in 81 kDa (VP1) and 56 kDa (VP2). The expressed proteins were then purified by affinity chromatography column. On the other hand, a CAV strain was used to infect MDCC-MSB1 cell line. After evaluated the virus titer, the CAV supernatant was inactivated by binary ethyleneimide. CAV inactivated virus coupled VP1 and VP2 were conducted to animal trial. The experimental groups included inactivated virus group, VP1/VP2 recombinant protein group, and inactivated viruses coupled recombinant protein group. Animal experiments shown vaccination the CAV inactivated virus coupled VP1 and VP2 group or VP1 plus VP2 group had mild symptoms after challenge. Therefore, the two groups had better immune efficacy and are potentially for developing a CAV subunit vaccine.

並列關鍵字

Chicken anemia virus ； subunit protein ； inactivated virus

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