新型豬輪狀病毒疫苗之研發

豬輪狀病毒（Porcine rotavirus; PRV）屬於呼腸孤病毒科，為一無外套模雙股RNA病毒，而豬輪狀病毒Ａ群（Porcine Rotavirus Group A; RVA）, 為誘發全世界幼齡動物病毒性下痢的重要病原之一。在養豬場中常造成豬隻大規模的下痢、生長性能差、甚至脫水死亡，本病毒可在1至8週齡的仔豬中檢測到，但因臨床症狀不易與其他病原區分而增加診斷和治療的成本，而造成嚴重的經濟損失。本研究目的在於開發出豬輪狀病毒的次單位疫苗並探討其最佳抗原量產的方法及免疫效力。我們選殖豬輪狀病毒具有中和抗體能力的VP7蛋白基因，使用桿狀病毒表現系統表現其蛋白並同時利用TCID50及即時定量反轉錄聚合酶鏈鎖反應(qRT-PCR)來偵測及定量病毒。最後搭配佐劑製成豬輪狀病毒疫苗進行安全及效力試驗以評估本疫苗的有效性。本研究以桿狀病毒表現系統生產豬輪狀病毒VP7蛋白之產量可達92 μg/mL，而病毒經MA-104細胞馴化培養16代後其病毒量可達108 TCID50/ml。以每劑量混合50μg VP7蛋白和106 TCID50的豬輪狀病毒添加佐劑製成疫苗免疫豬隻，ELISA試驗於免疫後1週即有上升的效果，於第6週達到高峰。因此本研究開發以桿狀病毒表現系統生產之蛋白結合經細胞馴化之病毒混和製成的豬輪狀病毒疫苗，仍保有抗原性可產生良好的中和抗體力價，深具發展疫苗之潛力。

關鍵字

豬輪狀病毒；下痢；昆蟲桿狀病毒； VP7 ；次單位疫苗

並列摘要

Porcine Rotavirus (PRV) belongs to the Reoviridae family and it is a double-stranded RNA virus without envelope. The Porcine Rotavirus Group A (RVA) is one of the most important pathogens for the viral diarrhea of young animals in the world. In pig farms, they often trigger large-scale swine diarrhea, poor growth performance, and even dehydration death. The virus can be detected in piglets between 1 and 8 weeks of age. However, because clinical symptoms are not easily distinguishable from other pathogens, it will increase the cost of diagnosis and treatment, resulting in serious economic losses. The objective of this study was to develop a new type vaccine for porcine rotavirus, exploring the optimal antigen production by evaluating the immune efficiency. We have selected PRV VP7 protein genes that have the capabilities of inducing neutralizing antibody, using the baculovirus expression system to express its proteins. TCID50 and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detected and quantified the virus. Finally PRV vaccine was prepared with adjuvants for safety and efficacy test to evaluate the effectiveness of the vaccine. In this study, the production of PRV VP7 protein can reach up to 92 μg/mL with the baculovirus expression system, and the virus titer can reach up to 108 TCID50/ml after sixteen generations of MA-104 domestication. Pigs were immunized with the vaccine mixed with the 50 μg of VP7 protein and 106 TCID50 of PRV. The ELISA test result showed an increased efficacy one week after immunization, and peaked at week 6. Therefore, the present study developed a porcine rotavirus vaccine, with the proteins produced by the baculovirus expression system, mixing with inactivated virus, with the aims of retain its antigenicity for enhanced protection and furthering the development of the vaccine.