- 學位論文
豬繁殖與呼吸道症候群次單位與不活化混合疫苗之研發
Development of inactivated and subunit vaccine against of Porcine reproductive and respiratory syndrome
摘要
豬繁殖與呼吸道症候群(Porcine reproductive and respiratory syndrome; PRRS)為世界上廣泛發生的豬隻傳染疾病。本病於1987年在北美首次被發現。其病原體豬繁殖與呼吸道症候群病毒(Porcine reproductive and respiratory syndrome virus; PRRSV)為一種具有外套膜的正股RNA病毒，屬於動脈病毒屬家族。PRRS透過感染豬隻鼻黏膜的上皮細胞以及肺部的巨噬細胞、單核球細胞，使得豬隻的免疫系統受到破壞，易引起繼發性感染。該病毒感染會引發豬隻食慾不振、高燒不退、腹瀉等臨床症狀，懷孕母豬被感染更會造成流產、產下木乃伊胎等，因此每當爆發疫情大流行時皆造成重大的經濟損失。PRRSV具有三種主要的結構蛋白包含核蛋白(N)、膜蛋白(M)、封套蛋白(GP5)，其中M 和GP5蛋白緊密結合併在病毒顆粒中形成雙硫鍵連接的異二聚體形式可誘導出中和抗體。本研究目的在開發PRRS不活化與次單位疫苗並制定最佳免疫程序。本研究以MARC-145細胞培養PRRSV經14代馴化後其病毒量達108.7TCID50/mL，且目前已成功轉殖M-GP5基因至pBacPAK8載體上，並透過桿狀病毒表達系統表現蛋白，經由SDS-PAGE及Western blot進行蛋白質分析及定量，結果顯示蛋白表現量可達到400µg/mL。動物試驗方面，小鼠試驗以不活化疫苗(108TCID50/mL)及次單位疫苗(50µg M-GP5蛋白)及不活化與次單位混合進行試驗，次單位及不活化與次單位混合之抗體力價皆高於不活化疫苗。小鼠試驗後採用其結果進行豬隻試驗，使用不活化疫苗(108TCID50/mL)及不活化與次單位(100µg M-GP5蛋白)混合疫苗進行試驗，並觀察豬隻抗體免疫反應。結果顯示，不活化疫苗之抗體力價仍高於不活化與次單位混合疫苗，但並無明顯差異，期望日後具有發展疫苗之潛力。
並列摘要
Porcine reproductive and respiratory syndrome (PRRS) is a worldwide porcine disease. It was originally identified in North America in 1987. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped positive-stranded RNA virus belonging to the Arteriviridae family. PRRS can infect pigs through nasal epithelium, pulmonary macrophages and monocytes, resulted in immunosuppression, which makes swines vulnerable to secondary infections. PRRS’s clinical symptoms are inappetence, fever, diarrhea and the birth of mummified piglets by farrowing sows, leading to significant economic loss. The virus contains three major structural proteins, including nucleocapsid protein (N), membrane protein (M), envelope glycoprotein (GP5), M and GP5 proteins are closely associated and formed disulfide-linked heterodimers in the virus particle, which can induce neutralizing antibodies. In this study, we will develop inactivated and subunit vaccine, and optimal immunization program for PRRS. Currently, PRRSV could be cultured in MARC-145 to reach108.7TCID50/mL after 14 passages. The M-GP5 genes were successfully cloned on the pBacPAK8 vector and expressed protein using the baculovirus expression system, Afterwards, SDS-PAGE and Western blot were undertaken to confirm and quantitate recombinant M-GP5 protein. These results showed that the expression of protein reached up to 400µg/mL. In an animal test, inactived vaccine(108TCID50/mL) and subunit vaccine(50µg M-GP5 protein), inactivated combine subunit vaccine used to immunize mice. The specific antibody potency of the subunit vaccine and inactivated combine subunit vaccine are significantly higher than inactived vaccine. After the mouse experiment, using inactived vaccine(108TCID50/mL) and inactivated combine subunit vaccine(100µg M-GP5 protein)to immunize pig, observe the antibody immune response of the pig. The results show that the antibody titer of the inactivated vaccine is still higher than inactivated and subunit vaccine, but there is no significant difference. It is expected that the vaccine will have the potential to develop in the future.