出版年:

2001

研究生:

王祥宇

研究生(英文姓名):

Shian-Yiu Wang

論文名稱:

假性狂犬病毒攜帶豬瘟病毒套膜蛋白重組病毒之構築

英文論文名稱:

Construction of recombinant pseudorabies viruses expressing E glycoproteins of classical swine fever virus

指導教授:

蕭璦莉

指導教授(英文姓名):

Ai-Li Shiau

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46881018

學年度:

89

語文別:

英文

論文頁數:

57

關鍵詞:

假性狂犬病毒 ; 豬瘟 ; 疫苗

英文關鍵詞:

pseudorabies ; CSFV ; vaccine ; PrV

被引用次數:

0

[ 摘要 ]

豬瘟病毒對於豬的畜牧產業常造成重大損失，因此早期即發展出具相當效率的減毒活性疫苗病毒株，但全病毒疫苗的使用無法區分野生病毒的感染及疫苗免疫的豬隻，因此疫苗的使用造成許多限制，故發展有效之豬瘟病毒可辨識疫苗是現階段研究的重點。由先前的研究報告指出，豬瘟病毒套膜蛋白為免疫系統產生有效中和抗體及細胞性免疫反應的目標，因此，本研究主要利用反轉錄聚合連鎖反應的方法將豬瘟病毒的全部外套膜蛋白（包含三個小次單位）選殖出來，接在大鼠β-actin 啟動子後面由其進行驅動轉譯蛋白質的表現而成為基因疫苗，並利用一gE、Tk基因缺損的假性狂犬病毒作為疫苗攜帶載體，此載體為本實驗室中所開發一快速篩選系統，當由大鼠β- actin 啟動子所驅動的豬瘟病毒外套膜蛋白基因被置換進入原假性狂犬病毒gD基因的位置時，可利用抗病毒藥物－Acyclovir 進行篩選，而得到純的重組病毒，命名為PrV-SY1。此重組假性狂犬病毒攜帶了可在哺乳類細胞表現的豬瘟病毒基因疫苗，並且由於gD基因的缺損造成此病毒載體只可造成一次的感染，無法在體內再製造出具感染力的病毒。在小鼠的實驗中發現，此重組病毒確實能同時引起抗豬瘟病毒和假性狂犬病毒的免疫反應。此試驗的成功，證明此假性狂犬病毒快速篩選載體表現系統的可行性。

[ 英文摘要 ]

Classical swine fever virus (CSFV) caused large damage in domestic industry. Therefore, effective live attenuated vaccine had developed at early. Though attenuated live virus vaccine induce high-titer neutralizing immune responses, it is difficult to distinguish the vaccination and wild-type virus infected pigs. Therefore to develop a marker vaccine and an epitope-specific serological test to differentiate between vaccinated and infected pigs is crucial for current CSFV vaccine development. In this study, we cloned genes envelope (E) encoding three subunits of the protein of CSFV, by RT-PCR. The cloned CSFV-E gene was inserted into a eukaryotic expressing vector under the control of the rat β-actin promoter to generate a genetic vaccine. This expression module was then inserted in place of the gD gene of a gE/thymidine kinase (TK)-negative Pseudorabies vector to generate a recombinant virus, which was designed PrV-SY1. By using a simple two-step method based on the herpes simplex virus thymidine kinase selection system, recombinant PrV deficient in gD, TK and gE, but carrying foreign genes can be efficiently constructed. Through serial acyclovir selection, the wild-type virus was killed and the recombinant PrV-SY1 was produced. Due to lack of the gD protein, the recombinant virus could not generate infectious progeny after administration. In the mouse immunization test, the PrV-SY1 could elicit high anti-CSFV and anti-PrV antibody responses. In conclusion, this recombinant virus may be further explored for developing an ideal vaccine in the future.