出版年:

研究生:

劉嘉雯

研究生(英文姓名):

Chia-Wen Liu

論文名稱:

發展假性狂犬病毒載體應用在重組疫苗及基因治療上

英文論文名稱:

Developement of pseudorabies viral vectors for recombinant vaccines and gene therapy

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:

S46861123

學年度:

87

語文別:

中文

論文頁數:

55

關鍵詞:

假性狂犬病毒 ; 假性狂犬病 ; 重組疫苗 ; 基因治療 ; 病毒載體

英文關鍵詞:

Pseudorabies virus ; Pseudorabies ; recombinant vaccines ;
gene therapy ; viral vectors

被引用次數:

1

[ 摘要 ]

假性狂犬病毒(pseudorabies virus, PRV)，隸屬於疹病毒科 (Herpesviridae)，引起豬隻假性狂犬病(pseudorabies, PR)，對養豬事業構成嚴重威脅。本病毒基因可製造多種醣蛋白，這些醣蛋白影響病毒複製的生活史，目前已知至少有七種以上的醣蛋白? gE (gI)、gB (gII)、gC (gIII)、gD (gp50)、gI (gp63)、gH (gH)、gG (gX)，這些醣蛋白與單純疹病毒的醣蛋白有很高的相似性。其中gE (gI)醣蛋白不是病毒複製所必須，所以常被拿來當標幟疫苗的指標，區分抗體的產生是因野外型感染或施打疫苗所引起。而醣蛋白gD (gp50)為病毒複製所必須，且與病毒的穿透力有關，但不會影響病毒在細胞與細胞間的擴散，當gp50缺乏時，病毒可藉著細胞與細胞間的傳遞達到局部擴散，由感染細胞所釋放出來的病毒子代，由於缺乏醣蛋白gp50，所以沒有感染性。
基於gp50的這些特性，本論文的目的為構築缺陷假性狂犬病毒應用在重組疫苗及基因治療上。我們重組了一個質體pBT/gp50 deletion，特性為具有胸腺核激(thymidine kinase, TK)基因，且它的兩旁序列與PRV醣蛋白gp50旁的序列相同，將此質體與R23 (PRV/gI-/TK-)雙基因缺損病毒株做同源性重組，在HAT培養液的篩選下，可得到重組病毒CW1 (PRV/gI-/gp50-/TK+)。由於gp50醣蛋白為PRV的必須基因，所以CW1病毒必須在包裝細胞NIH3T3/gp50中才可複製。此病毒載體不僅具有活毒疫苗的特性；也是個標幟疫苗，在活體試驗下證實，此病毒載體的確可抵抗PRV的感染，對小鼠具有保護力。
以相同的原理，將質體pBT/gp50 deletion的TK基因，以基因工程的方式換成增強型綠色螢光蛋白(enhanced green fluorescent protein, EGFP)。改成EGFP的目地為EGFP為自發性螢光，不需外加其它物質即可觀察，且表現穩定，是一個很好的報告基因。將此質體pBT/EGFP與病毒CW1 (PRV/gI-/gp50-/TK+)做同源性重組，在acyclovir的篩選下，可以得到重組病毒CW2 (PRV/gI-/gp50-/TK-/EGFP+)。利用CW2病毒帶有標幟基因EGFP，可以評估此系統的運作及應用在基因治療上的可行性。
建立此系統的好處在於：任何有興趣的基因，利用重組DNA的方式，取代原來的質體pBT/gp50 deletion中的TK基因，再與CW1病毒進行同源性互換，在acyclovir的篩選下，可以快速得到重組假性狂犬病毒，進而應用在重組疫苗及基因治療上！

[ 英文摘要 ]

The envelope glycoprotein gp50 of pseudorabies virus (PRV) is essential for virus entry, but is not required for subsequent steps in the viral replication cycle. Phenotypically-complemented gp50 null mutants can infect cells and can spread, both in vitro and in vivo, by direct cell-to-cell transmission. However, progeny virions released by the infected cells are non-infectious because they lack gp50. The aim of this study is to construct gp50-defective PRV vectors for the applications of recombinant vaccines and gene therapy. The transfer vectors carrying the genes encoding either herpes simplex virus thymidine kinase (HSV-TK) or enhanced green fluorescent protein (EGFP) were designed to replace the gp50 gene. By homologous recombination, NIH3T3 cells expressing PRV gp50 were cotransfected with the transfer vector and the TK-/gI- PRV that is defective in both viral TK and gI, a nonessential glycoprotein. The recombinant PRV carrying HSV-TK, but defective in gp50 and gI, was isolated by selection with hypoxanthine, aminopterin and thymidine (HAT) media, and the HSV-TK gene was also detectable by PCR in the viral supernatant. The plaque size produced by gp50-defective PRV was bigger than that produced by parental TK-/gI- PRV. Moreover, the former was more sensitive than the latter to acyclovir. Mice treated intraperitoneally with gp50-/HSV-TK+ or parental TK-/gI- PRV have prolonged survival time compared with those treated with wild-type PRV. These results suggest that gp50-defective PRV may be exploited for the development of recombinant PRV vaccines. A similar strategy was used to replace the HSV-TK gene with the EGFP gene following acyclovir selection. The EGFP gene was detected by PCR in the viral supernatant. The expression of EGFP in NIH3T3/gp50 cells infected with TK-/gI-/gp50-/EGFP+ virus was examined using a standard fluorescein isothiocyanate (FITC) filter-equipped fluorescence microscope. Taken together, the gp50-defective PRV may be explored as potential mammalian expression vectors for recombinant vaccines and for gene therapy.