臺灣博碩士論文加值系統

豬第二型環狀病毒（PCV2）主要引起生長豬隻之離乳後多系統性消耗症（PMWS），並容易導致二次性感染而死亡。病毒之基因序列包含11個開放讀碼區（ORF），其中ORF 1轉譯為病毒之複製相關蛋白（replication-associated proteins），ORF 2則轉譯為核衣蛋白（Capsid protein）。本試驗應用大腸桿菌進行PCV2 ORF 1與ORF 2基因片段之表現，並與昆蟲細胞所表現之ORF2蛋白進行動物免疫效果比較。同時，於ORF 2基因序列之5’端連接去毒性之綠膿桿菌外毒素A（以PE407或PE532表示）基因序列，以及（或）3’端加入KDEL氨基酸（以K表示）之基因序列，並於大腸桿菌進行表現。本試驗成功表現出21 KDa的PCV2ORF1、54 KDa的PCV2ORF2、56 KDa的PCV2ORF2K、81 KDa的PE407-ORF2、100 KDa的PE407-ORF2K、95 KDa的PE532-ORF2與97 KDa的PE532-ORF2K等重組或嵌合蛋白。昆蟲細胞表現系統則表現出34 KDa的Bac-PCV2ORF2重組蛋白。將上述重組蛋白進行豬隻與小白鼠之免疫試驗後發現，PCV2ORF1、PCV2ORF2、PE407-ORF2K與Bac-PCV2ORF2等均可誘發出PCV2抗體，其中以Bac-PCV2ORF2所誘發的PCV2抗體力價最高。這些重組蛋白可作為未來開發PCV2次單位疫苗之用。

Porcine circovirus type 2 (PCV2) has been reported to be associated with the development of porcine multisystemic wasting syndrome (PMWS), which often results in the death of pigs due to secondary infection. The genome of PCV2 might contain eleven open reading frames (ORFs). ORF 1 and ORF 2 encode the replication-associated proteins and capsid protein, respectively. In this study, the immunogenicity of E. coli-expressed PCV2 ORF 1 and PCV2 ORF 2 and baculovirus-expressed PCV2 ORF 2 were compared in mice and pigs. In addition, ORF 2 gene with Pseudomonas aeruginosa exotoxin A (expressed as PE407 and PE532) gene added at 5’ terminal, and/or the KDEL amino acids gene (expressed as K) added at 3’ terminal were constructed and expressed in E. coli. A total of seven recombinant proteins including PCV2 ORF1 (21 KDa), PCV2 ORF 2 (54 KDa), PCV2 ORF2K (56 KDa), PE407-ORF2 (81 KDa), PE407-ORF2K (100 KDa), PE532-ORF2 (95 KDa) and PE532-ORF2K (97 KDa) were successfully expressed in E. coli. A recombinant protein of 34 KDa (Bac-PCV2ORF2) was also expressed in baculovirus. Antibodies against PCV2 could be detected when pigs or mice were immunized with PCV2ORF1, PCV2ORF2, PE407-ORF2K and Bac-PCV2ORF2. Among the recombinant proteins tested, Bac-PCV2ORF2 induces the best antibody titer in animals. The aforementioned recombinant proteins can be used as the components for the development of PCV2 subunit vaccine in the future.

中文摘要………………………………………………………………… Ⅰ
英文摘要………………………………………………………………… Ⅲ
誌謝……………………………………………………………………… Ⅳ
目錄……………………………………………………………………… Ⅴ
圖次…………………………………………………………………...….. XI
表次………………………………………………………………………. XⅢ
第一章 緒言……………………………………………………………… 1
第二章 文獻回顧……………………………………………………… ... 3
2.1 豬環狀病毒之病毒特行………………………………………..…. 3
2.1.1 病毒結構與理化特性………………………………………….. 3
2.1.2 病毒複製……………………………………………………….. 3
2.1.3 基因體分析…………………………………………………….. 5
2.2 豬第二型環狀病毒之流行病學…………………………………... 7
2.3 臨床症狀…………………………………………………………... 9
2.4 病理變化…………………………………………………………... 10
2.4.1 肉眼病變……………………………………………………….. 10
2.4.2 組織病理學…………………………………………………….. 11
2.5 PCV2感染對免疫系統之影響……………………………………. 12
2.5.1 PCV2感染對血液學之影響…………………………………… 12
2.5.2 PCV2感染對組織中免疫細胞之影響………………………… 14
2.5.3 PCV2感染對樹狀突細胞與巨噬細胞之影響………………… 17
2.5.4 PCV2感染對細胞凋亡之影響………………………………… 19
2.5.5 PCV2感染對細胞激素之影響………………………………… 19
2.5.6 亞麻油酸對PCV2所引發的免疫抑制之影響…………..…… 20
2.6 豬隻受PCV2感染後之致病機轉… … .………………………… 21
2.7 PCV2之診斷………………………………………………………. 22
2.8 PCV2之控制………………………………………………………. 24
第三章 材料與方法……………………………………………………… 26
3.1 病毒來源……………………………………………………….….. 26
3.2 細胞來源……………………………………………………….….. 26
3.3 病毒分離……………………………………………………….….. 27
3.4 以IFA進行PCV2病毒檢測………………………………….….. 28
3.5 PCV2病毒核酸萃取…………………………………………...….. 28
3.6 以PCR檢測PCV2……………………………………………….. 29
3.7 以即時定量聚合酶鏈反應（real-time PCR）進行定量…… ….. 30
3.7.1 PCV2之病毒定量………………………………………… ……30
3.7.2 PRRSV之病毒定量……………………………………… ..….. 32
3.8以IFA進行抗體檢測…………………………………….…………33
3.8.1 PCV2抗原盤之製作………………………………….…………33
3.8.2 PRRSV抗原盤之製作……………………………….………….33
3.8.3 間接螢光抗體檢測………………………………….……. ……34
3.9 表現質體之構築與選殖……………………………….……... ….. 34
3.9.1 增幅PCV2及PE之基因片段…………………….……….… 35
3.9.1.1 增幅PCV2 ORF1基因片段…………………….… ..….… 35
3.9.1.2增幅PCV2 ORF2之基因片段………………….……….… 37
3.9.1.3增幅PCV2 ORF2K之基因片段……………….………..… 38
3.9.1.4增幅PE之基因片段…………………………….……….… 40
3.9.2 載體之前處理……………………………………….… ..….… 41
3.9.3 PCR產物之純化以及限制酶之切割……………….………… 42
3.9.4 接合作用…………………………………………….….… ..… 42
3.9.4.1 pRSETa-PCV2ORF1、pET32a-PCV2ORF2、pET32a-PCV2 ORF2K、pRSETa-PE407、pRSETa-PE532及pBlueBac
His2a- ORF2之接合作用……………….………………… 42
3.9.4.2 pRSETa-PE407-ORF2、pRSETa-PE532-ORF2、pRSETa-
PE407- ORF2K與pRSETa-PE532-ORF2K之接合作用... 43
3.9.4.3 pET32a-PE407-ORF2K之接合作用.……………………… 43
3.10 轉形作用………………………………………………………… 43
3.11 菌落篩選…………………………………………………….……44
3.11.1 小量質體之萃取………………………………………………44
3.11.2 限制酶切割…………………………………………… ...……45
3.11.3 核酸定序………………………………………………………45
3.11.4 大量質體之萃取………………………………………………45
3.12 以大腸桿菌（E. coli）進行蛋白質表現…………………….… 47
3.12.1 轉形作用………………………………………………… .… 47
3.12.2 小量蛋白質表現………………………………………………47
3.12.3 大量蛋白質表現………………………………………...…….48
3.13 以桿狀病毒（Baculovirus）進行蛋白質表現………………….48
3.13.1 基因重組桿病毒（recombinant baculovirus）之製備………49
3.13.2 重組桿狀病毒之純化……………………………………...….50
3.13.3 重組桿狀病毒之確認……………………………………...….50
3.13.4 以SF9細胞進行蛋白質表現…………………………………51
3.14 蛋白質之純化…………………………………………………….51
3.14.1 不可溶蛋白之純化……………………………………….…...52
3.14.1.1 管柱製備…………………………………………………...51
3.14.1.2 蛋白質純化……………………………………………….. 52
3.14.1.3 蛋白質復性……………………………………………….. 52
3.14.2 可溶性蛋白之純化……………………………………………53
3.14.2.1 管柱製備………………………………………………….. 53
3.14.2.2 蛋白純化………………………………………………….. 53
3.15十二烷基硫酸鈉聚丙烯醯胺電泳………………………………. 53
3.16 西方轉漬法………………………………………………….……54
3.17 動物試驗…………………………………………………….……54
3.17.1 實驗動物………………………………………………………54
3.17.2 抗原製備………………………………………………………55
3.17.3動物試驗1：豬隻以E. coli所表現之PCV2及PRRSV
蛋白接種後之免疫反應….………………………………. … 55
3.17.4動物試驗2：小白鼠以E. coli或Baculovirus所表現之
PCV2或PRRSV蛋白接種後之免疫反應…………………. 56
3.17.5動物試驗3：豬隻以E. coli或Baculovirus所表現之
PCV2蛋白接種後之免疫反應..……………………………...57
3.17.6動物試驗4：小白鼠以以E. coli或Baculovirus所表現
之PCV2或PRRSV蛋白接種後之免疫反應.……………….58
3.18 統計分析…………………………………………………….…... 59
第四章 結果………………………………………………………………. 60
4.1應用大腸桿菌表現系統表現PCV2及PRRSV蛋白…………… 60
4.1.1表現質體之構築………………………………………….……. 60
4.1.1.1 利用PCR增幅PCV2 ORF1、PCV2 ORF2、PCV2 ORF
2K、PE407 與PE532基因片段…………………………60
4.1.1.2 表現質體之選殖………………………….…………………60
4.1.2重組蛋白之表現……………………………….………………..61
4.2應用昆蟲細胞表現系統表現PCV2及PRRSV蛋白.……………62
4.2.1 表現質體之構築………………………………….……….……62
4.2.2 基因重組桿狀病毒之篩選…………………….……….………62
4.2.3 重組蛋白之表現……………………………….……….………63
4.3 動物試驗1之結果……………………………….………..………63
4.3.1 抗體力價變化及病毒檢測…………………….……….………63
4.3.2 臨床症狀……………………………………….……….………64
4.3.3 肉眼病變……………………………………….……….………64
4.3.4 組織病理學…………………………………….……….………. 64
4.4動物試驗2之結果………………………………….……….………65
4.5動物試驗3之結果………………………………….……….………65
4.6動物試驗4之結果………………………………….……….………65
4.6.1 PCV2抗體力價之變化………………………….…… ..………. 66
4.6.2 PRRSV抗體力價之變化……………………….………………. 66
第五章 討論……………………………………………….….………..…. 67
第六章 參考文獻………………………………………………………… 100
第七章 附錄……………………………………………………………… 114
7.1 PCV1與PCV2之stem-loop序列與結構圖……….………………114
7.2 PCV1（np）與PCV2（pmws）基因序列中poly（A）與CGGC
AG motif之位置……….………………………………………….. 115
7.3 PCV1（PK-15）與PCV2（Stoon）之ORF 1 motif及P-loop
區域…………………………………………………….………… 116
7.4 pRSETa載體結構圖…………………………………………….… 117
7.5 pET32a載體結構圖……………………………………………….. 118
7.6 各動物試驗所使用之免疫抗原表……………………………….. 119
7.7 Bac-PRRSVORF5與Bac-PRRSVORF6之SDS-PAGE及西
方轉漬圖……………………………………………………..…….. 120
7.8溶液的配製.…………………………………………………...….. 121
作者簡介………….………………………………………………………. 129

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