臺灣博碩士論文加值系統

豬瘟是由豬瘟病毒感染所引發之高度傳染性動物疾病，一旦爆發將會導致非
常嚴重的經濟損失。豬瘟病毒封套膜醣蛋白 E2 已經被證實可在豬隻體內誘發具
有保護力之中和抗體，本研究即應用酵母菌表現系統進行豬瘟病毒封套膜醣蛋白
E2 之大量表現並測定其疫苗效力。由於蛋白產量會受到胺基酸密碼子使用率之影
響，遂將 E2 醣蛋白之第一個胺基酸密碼子 (CGG) 修改為(AGA) 以提高使用效
率，亦移除蛋白之穿膜區域進而構築出三種不同長度的 E2 重組表現蛋白並分別
命名為 N342 、 N330 及 N301。經密碼子修改後之表現蛋白其產量有明顯提升，
且免疫三種不同 E2 重組蛋白之各組豬隻在補強免疫後皆可測得 E2 特異性抗體
及具保護能力之中和抗體力價且持續 14 週之久。豬隻免疫實驗之結果顯示酵母
菌所生產之 E2 重組表現蛋白 N342 、 N330 及 N301 均具有做為次單位疫苗之
潛力。另外，豬瘟病毒醣蛋白 Erns 在病毒感染細胞的過程中主要參與病毒與細胞
表面heparan sulfate 的結合，具有吸附的功能。本研究利用酵母菌表現系統及全
基因密碼子優化所生產之全長 Erns (N227) 及C 端缺損片段 (N190) 皆具有與
heparin 管柱結合之能力，推測除了C 端之外仍有其它的heparin 結合位置。進
一步進行豬瘟病毒 Erns 醣蛋白胺基酸序列比對分析後發現在N 端胺基酸編號
34 到 44 區域內可能具有一個heparin binding domain (HBD)，將其中編號 36
(K36) 及 39 (K39) 之鹼性胺基酸lysine (K) 進行點突變為 alanine (A) 後其對於
heparin 管柱之結合能力有下降趨勢且與細胞表面之結合能力亦顯著下降，進一步
突變編號 43 (T43) 及 44 (H44) 則有輕微的影響。證明豬瘟病毒 TD96 病毒株之
Erns 蛋白 N 端區域帶有一個新發現的heparin binding domain ，而其中之鹼性胺
基酸K36 及 K39 扮演著影響蛋白與 heparin 結合力之關鍵角色。

Classical swine fever virus (CSFV) infection is a severe swine disease, often
causing large economic losses. Pichia pastoris yeast-expressed CSFV glycoprotein E2
(yE2) has been shown to induce a protective immune response against the virus. To
improve the expression level of yE2, the first codon of E2 gene (CGG) was optimized
to the most favorite codon (AGA) and deleted transmembrane domain. Three
recombinant E2 glycoproteins N342, N330 and N301 which composed the N-terminal
342, 330 and 301 residues respectively, were constructed. The yield of E2 protein was
remarkably increased in the codon optimized strain (N342). The immunogenicity of
each recombinant E2 subunits was confirmed by the immunization of pigs, and all
immunized groups demonstrated high neutralizing antibody titers after boost
immunization, which lasted for a long period of time. In addition, the envelope
glycoprotein Erns has been shown to bind to the sulphated-heparin like
glycosaminoglycans (GAGs) on cell surface and participates in cell attachment of the
virus. The CSFV Erns gene was codon optimized for the expression in Pichia pastoris.
The C-terminally truncated Erns recombinant protein lacking the previously identified
heparin-binding domain (HBD) bound to heparin column, suggesting the presence of
another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed
a common potential N-terminal HBD at residues 34-44. Site-directed mutagenesis of the
basic amino acids at K36 and K39 significantly reduced the heparin-binding affinity of
the protein. Further mutations of both T43 and H44 had little effect. Thus, a novel
potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been
detected, and the two basic amino acids K36 and K39 are crucial for binding activity to
heparin matrix and cell-surface GAGs.

摘要....................................................................................................................................i
Abstract............................................................................................................................ii目次..................................................................................................................................iii
表次...................................................................................................................................v
圖次..................................................................................................................................vi
第一章 緒言.....................................................................................................................1
第二章 文獻探討.............................................................................................................3
第一節 豬瘟之歷史背景.........................................................................................3
第二節 豬瘟病毒結構與特性.................................................................................4
一、豬瘟病毒分類特性....................................................................................4
二、豬瘟病毒結構蛋白....................................................................................5
三、豬瘟病毒非結構蛋白................................................................................7
第三節 豬瘟病毒之生命週期.................................................................................9
一、 豬瘟病毒傳染途徑...................................................................................9
二、 豬瘟病毒致病機轉...................................................................................9
三、 豬瘟病毒之臨床症狀............................................................................9
第四節 豬瘟病毒之診斷技術...............................................................................10
一、 豬瘟病毒抗原之偵測.............................................................................10
二、 豬瘟病毒血清學之偵測.........................................................................11
第五節 豬瘟病毒之防疫......................................................................................11
一、 豬瘟疫苗種類.........................................................................................11
二、 世界防疫現況.........................................................................................12
三、 台灣防疫現況與未來展望.....................................................................13
第六節 硫酸乙醯肝素與肝素...............................................................................14
一、 肝素與硫酸乙醯肝素...........................................................................14
二、 Erns之肝素結合位..................................................................................15
第七節 酵母菌表現系統.......................................................................................15
一、 酵母菌表現系統簡介...........................................................................15
二、 密碼子優化 (codon optimization)........................................................16
第三章 材料與方法.......................................................................................................17
第一節 病毒與細胞及表現菌株...........................................................................17
一、豬瘟病毒株...............................................................................................17
二、豬瘟病毒 Erns 蛋白序列比對..................................................................17
三、細胞與表現菌株培養...............................................................................17
第二節 表現質體之構築與分析...........................................................................18
一、 引子對設計.............................................................................................18
二、 基因密碼子優化.....................................................................................18
三、 聚合酶連鎖反應增幅目標基因.............................................................18
四、 肝素結合位點突變 (site-directed mutagenesis)....................................19
五、 重組質體之構築與大腸桿菌之轉型作用 (transformation).................20
第三節 酵母菌之轉型作用與蛋白表現...............................................................20
一、 酵母菌轉型作用.....................................................................................20
二、 酵母菌表現重組蛋白.............................................................................22
三、 重組蛋白之保存.....................................................................................22
第四節 表現蛋白之特性分析...............................................................................22
一、 管柱純化分析........................................................................................22
二、 西方墨點法分析表現蛋白.....................................................................23
三、 Erns 蛋白之 RNase 活性分析.............................................................24
四、 Erns 重組蛋白與細胞表面結合力之測定.............................................24
五、 重組 Erns干擾豬瘟病毒感染測試........................................................25
第五節 動物免疫試驗...........................................................................................26
一、 豬隻免疫重組 E2 次單位疫苗............................................................26
二、 免疫後豬隻血清學測試.........................................................................26
第四章 結果...................................................................................................................28
第一節 豬瘟病毒醣蛋白 E2 之表現及疫苗效力測試......................................28
一、 豬瘟病毒醣蛋白 E2 之表現及分析....................................................28
二、 豬瘟病毒醣蛋白 E2 表現蛋白之疫苗效力測試................................29
第二節 豬瘟病毒醣蛋白 Erns 之表現及分析....................................................29
一、 豬瘟病毒醣蛋白 Erns 之表現及分析....................................................29
二、 Erns 重組蛋白片段與肝素管柱及細胞表面乙醯肝素結合之測定...31
三、 不同 Erns 重組表現蛋白干擾豬瘟病毒感染細胞之測定..................32
第五章 討論...................................................................................................................50
參考文獻.........................................................................................................................54
附錄.................................................................................................................................66

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