臺灣博碩士論文加值系統

豬環狀病毒第二型(porcine circovirus type 2, PCV2)屬於環狀病毒科，病毒核酸為環狀、單股DNA，基因組全長1.7 kb，其中的ORF2為病毒外鞘蛋白基因，是重要的抗原基因。但是本病毒的主要結構蛋白ORF2，在轉殖入大腸菌後進行原核表現後卻難以大量表現，因此，本論文先將PCV2 ORF2基因均分為三段分別進行表現，發現除了靠近N端的第一段(F1)外，其餘片段皆可大量表現。接著若將F1 + F2 (稱F6片段) 以及F2+F3 (稱F5片段) 片段分別選殖及表現，則靠近N端的F6片段無法表現，F5片段仍為高量表現。若將PCV2 ORF2基因均分為二，則如同預期的，靠近C端的F4片段可以大量表現。將PCV2 ORF2序列進行胺基酸分析，發現F1片段擁有21個精胺酸，遠多於F2+F3片段的9個。因此，另外分別構築了缺失前9、21、36、48、111個核苷酸的ORF2缺失片段，分別稱為F10、F22、F37、F49、F112片段再加以選殖表現，發現PCV2 ORF2各個重組蛋白之表現量隨著精胺酸基因密碼的增加而減少。將F5片段的重組蛋白經純化後接種大白兔，可以誘發高力價的抗體，該抗體可以IPMA法檢測出PK-15細胞內被PCV2感染之病毒抗原。基於目前尚無PCV2商業用疫苗問世，因此本論文也探討了開發PCV2次單位及DNA疫苗之可行性。以大鼠為模式，探討了F2、F3、F5、F49各重組蛋白所誘發之抗體情形，也比較了F5與F49重組蛋白併用含ORF1及ORF2 之真核表現重組載體分子或併用含ORF2單一真核表現重組載體分子免疫大鼠後之抗體上升之情形。除了以IPMA法測中和抗體力價外，同時以ELISA法比較各組血清抗體之呈現情形。ELISA的結果指出，以F2片段的重組蛋白免疫大白鼠後能夠誘發最高量的IgG抗體，但是中和抗體力價的結果顯示，不管是原核表現之重組蛋白或併用真核表現重組載體分子，若以大白鼠為動物模式，皆無法誘發高量中和抗體。

Porcine circovirus type 2 belongs to the Circoviridae family. PCV2 is a small, nonenveloped virus with a single-stranded circular DNA genome of about 1.76 kb. The open reading frame ORF2 of the PCV2 genome encodes the immunogenic structural capsid protein. The expression for this immunogenic protein using full length ORF2 DNA was difficult to obtain in large quantity in prokaryotic expression system. Therefore, an alternative approach was to divide ORF2 DNA into three segments for expression, F1, F2, and F3. It was discovered that expression in E.coli was obtained in segments other than F1, which contained the N-terminal sequences. When F1+F2 (named F6) and F2+F3 (named F5) fragments were cloned and expressed, F6, similar to F1, was not expressed due to the N-terminal sequences. On the other hand, F5 was still expressed in large quantity. When dividing the full length ORF2 into half, F4 with C-terminal sequences was again expressed in large quantity as predicted. Analysis on PCV2 ORF2 full length sequence showed that within F1, there were 21 arginine codons, far more than the 9 arginine codons in F2 and F3 combined. Therefore, five additional constructs with the first 9, 21, 36, 48, 111 nucleotides missing of ORF2 (named F10, F22, F37, F49, and F112 respectively) were cloned and subsequently expressed. It was discovered that PCV2 ORF2 recombinant protein expression increased as arginine codons decreased. When immunizing rat with purified F5 recombinant protein, high antibody titer against PCV2 was induced, as confirmed by the IPMA method in PCV2 infected PK-15 cells. Currently, since PCV2 vaccine is not commercially available, this research project was also aimed for the development of a PCV2 subunit vaccine. Rats were used as an experimental animal model to evaluate the various recombinant proteins, F2, F3, F5, and F49, for their abilities to induce antibodies against PCV2. In addition, F5 and F49 recombinant proteins, in conjunction with ORF1 and/or ORF2 recombinant eukaryotic expression plasmids, were injected in rats to evaluate their abilities to induce antibodies against PCV2. Other than using IPMA for the detection of neutralizing antibodies, I used ELISA to measure the levels of antibodies induced in the collected rat serum of different experimental groups. The result of ELISA indicated that F2 recombinant protein induced the highest level of IgG antibodies in rat. As for neutralizing antibodies, neither the recombinant proteins nor when used in conjunction with recombinant eukaryotic expression plasmids were induced when using rat as an animal model.

目錄

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中文摘要…………………………………………………………………….……….....i
英文摘要 …………………………………………………………………....…..….....ii
目錄…………………………………………………………………….....…………...iii
表目錄…………………………………………………………………...…………….vi
圖目錄……………………………………………………………………...…………vii

第一章 緒言………………………………………………………………………….1

第二章 文獻探討…………………………………………………………………….2

一、 豬環狀病毒的歷史…….…………….…..…………………………….….2
二、 豬環狀病毒的形態學及基因組成……….…...……………...……….…..2
三、 PCV2的診斷…………………....….……………...……………………....4
四、 PCV2重組蛋白的表現及研究………….………..…..………….…….…4
五、 原核表現系統的探討…………………….………...…………..................5
六、 重組蛋白的表現及純化………………………………..…………………6
七、 DNA疫苗的原理及其應用…...….…………...……………………..…....6

第三章 材料與方法……………………………………………………………….....8

一、 含PCV2 ORF1及ORF2之質體DNA製備…………….……….….......8
二、 設計可增幅不同長度PCV2 ORF2基因之引子.……...…………..….....8
三 、 構築含PCV2 ORF1及ORF2之真核表現重組載體……….…...…….....9
四、 以PCR法增幅PCV2各DNA片段……………….……….…...…….....9
五、 PCR產物的純化………………………………..……...............................9
六、 限制酶切割反應(Restriction Enzyme Cleavage Reaction)……………...10
七、 接合反應(Ligation)……………………………………………… ……..10
八、 勝任細胞(Competent Cell)的製備及轉型作用(Transformation)…........10
九、 重組質體的挑選及確認…………………………………………….…..11
十、 重組載體的定序………………………………………………………...11

ORF2各重組蛋白之表現及定性分析

一 、 以IPTG誘導大腸菌中重組蛋白的表現……………………....………..11
二、 SDS-PAGE確認各片段ORF2重組蛋白的表現……………..………...12
三、 西方墨漬法分析(Western Blot)….……………………….………….…..12

以大白鼠為模式評估ORF2各重組蛋白及PCV2 真核表現重組載體分子之免疫效果

一、 大量純化大腸桿菌中表現的ORF2重組蛋白……………….………….13
1. 確認重組蛋白表現的形態………..…………………………………13
2. 包涵體形態基因重組蛋白之純化…..………………………………13
3. 重組蛋白純化後的定量………………..……………………………14
二、 真核表現重組載體分子的製備……….……………………….…….…...14
三、 真核表現重組載體分子的轉染(Transfection)及定性分析……….….….15
四、 大白鼠的免疫…………………………………………………………….15
五、 PCV2 ORF2高免抗體的製備……………………………………………16
六、 大白鼠血清中和PCV2抗體力價的測定…….…...……………………..16
1. 細胞及PCV2種毒……………………………………....……..…….16
2. PCV2種毒力價的測試………………………...………………..…..16
3. 免疫過氧化酶單層細胞分析法(Immuno Peroxidase
Monolayer Assay, IPMA)…………….………...……………….….17
4. 大鼠血清中和抗體力價的測定………………………..….……......17
5. ELISA法檢測PCV2抗體IgG及IgM的力價………….………...17
實驗策略一……………………………………………………………………….19
實驗策略二……………………………………………………………………….20

第四章 結果…………………………………………………………………..…….21

ORF2基因的選殖、確認與表現

一、 以不同引子對分別增幅6段ORF2基因…………………..…………..21
二、 分段ORF2基因的選殖與確認………………………..………...…….. 21
三、 ORF2重組蛋白的表現及確認………………………..……….............. 21
四、 重新設計引子將ORF2全長做N端之漸近缺失……..………. ….…..22
五、 選殖N端漸近缺失的ORF2基因……………………..………….……22
六、 各ORF2 N端漸近缺失片段的表現…………………..…………….….22

以大白鼠為模式探討PCV2 ORF2重組蛋白及併用真核表現重組載體分子之免疫效果

一、 ORF2重組蛋白的純化及真核表現重組載體分子的製備…….………23
二、 真核表現重組載體分子於真核細胞內之表現....….……………….…...23
1. 以ORF1及ORF2/F5重組蛋白免疫白兔獲致抗PCV2
ORF1及ORF2之高免抗體………………………………………..23
2. 真核表現重組載體分子轉染PK15細胞後重組蛋白的表現…….24
三、 動物免疫及抗體力價分析…………………..…………….………..........24
1. 以ELISA法檢測大白鼠抗ORF2之抗體…………………….….24
2. 大白鼠血清中和抗體力價測試…………………………..……….25

第五章 討論………………………………………………………………………...26

附錄…………………………………………………………………………………...31

參考文獻……………………………………………………………………...............61




表目錄

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表一、 PCV2 ORF2基因進行分段表現所使用之引子序列及其PCR
產物預期長度和各重組蛋白之預估分子量…………………..………………31
表二、 PCV2 ORF2以PCR法將PCV2 ORF2基因5’端進行不同長度
缺失突變，所使用之引子序列及其PCR產物預期長度及各重
組蛋白預估分子量…………………………………………………………..31
表三、 以PCR法將PCV2 ORF1及ORF2選殖入真核表現載體pcDNA3
所使用之引子序列及其PCR產物長度……………………………………32
表四、 PCV2 ORF2胺基酸序列中精胺酸之數量分析表…………………………32




















圖目錄

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圖一、 各種環狀病毒的複製起始點序列之比較…………………………………..33
圖二、 PCV1和PCV2基因組的比較圖……………………………………………34
圖三、 原核表現系統載體pET24a……..…………….…………………………….35
圖四、 掃瞄式電子顯微鏡下的包涵體……………………………………………..35
圖五、 DNA疫苗的作用原理………………………………………………………36
圖六、 真核表現載體pcDNA3 …………………………………………………….37
圖七、 ORF2分段表現示意圖……………………………………………………...37
圖八、 以真核表現重組載體分子及各重組蛋白接種大白鼠之免疫流程……….38
圖九、 分別以不同引子對進行PCR，增幅PCV2 ORF2之各DNA片段………39
圖十、 將PCV2 ORF2/F1片段選殖入原核表現載體pET24a後進行DNA
定序之結果………………………………………………………………….40
圖十一、 將PCV2 ORF2/F2片段選殖入原核表現載體pET24a後進行DNA
定序之結果……………………………………………………………….40
圖十二、 將PCV2 ORF2/F3片段選殖入原核表現載體pET24a後進行DNA
定序之結果……………………………………………………………….41
圖十三、 將PCV2 ORF2/F4片段選殖入原核表現載體pET24a後進行DNA
定序之結果……………………………………………………………….41
圖十四、 將PCV2 ORF2/F5片段選殖入原核表現載體pET24a後進行DNA
定序之結果……………………..………………………………………...42
圖十五、 將PCV2 ORF2/F6片段選殖入原核表現載體pET24a後進行DNA
定序之結果……………………………………………………………….42
圖十六、 以大腸桿菌分別表現PCV2 ORF2各片段之重組蛋白………………..43
圖十七、 以西方墨漬法檢測PCV2 ORF2各片段重組蛋白之表現……………..44
圖十八a、 PCV2 ORF2/F1片段胺基酸序列中精胺酸位置的分佈情形…….…45
圖十八b、 設計漸近缺失突變的PCV2 ORF2各引子F10、F22及F37之
位置圖…………………………………………………………………...45
圖十八c、 設計漸近缺失突變的PCV2 ORF2各引子F49、F112及F154位
置圖……………………………………………………………………...45
圖十九、 以PCR法將PCV2 ORF2基因5’端進行不同長度缺失突變之結
果分析圖……………………………………………………………….…46
圖二十、 將PCV2 ORF2/F10片段選殖入原核表現載體pET24a後進行
DNA定序之結果………………………………………………………...47
圖二十一、 將PCV2 ORF2/F22片段選殖入原核表現載體pET24a後進行
DNA定序之結果……………………………………………………...48
圖二十二、 將PCV2 ORF2/F37片段選殖入原核表現載體pET24a後進行
DNA定序之結果……………………………………………………...49
圖二十三、 將PCV2 ORF2/F49片段選殖入原核表現載體pET24a後進行
DNA定序之結果……………………………………………………...50
圖二十四、 將PCV2 ORF2/F112片段選殖入原核表現載體pET24a後進行
DNA定序之結果……………………………………………………...51
圖二十五、 將PCV2 ORF2/F154片段選殖入原核表現載體pET24a後進行
DNA定序之結果……………………………………………………...52
圖二十六、 PCV2 ORF2/F154變異片段eF154胺基酸序列分析圖……………..53
圖二十七、 以大腸桿菌分別表現經過缺失突變後的各PCV2 ORF2片段…..…54
圖二十八、 以西方墨漬法檢測PCV2 ORF2各片段重組蛋白之表現…………..55
圖二十九、 以SDS-PAGE分析經純化之PCV2 ORF2各重組蛋白…………….56
圖三十、 PCV2 ORF1及ORF2選殖入真核表現載體pcDNA3之DNA
電泳分析圖…………………………………………………………….....57
圖三十一、 以PCV2 ORF2/F5重組蛋白免疫白兔製備高免抗體的定性
分析圖……………………………………………………………….....58
圖三十二、 以IPMA法分析真核表現重組載體分子在PK15細胞中的
表現情形…………………………………………………………….....58
圖三十三、以ELISA法檢測ORF2各重組蛋白及真核表現重組載體分
子免疫大白鼠後各組血清IgG抗體之分析圖……………………..….59
圖三十四、 以Jameson and Wolf程式預測PCV2 ORF2胺基酸序列之
抗原性指標分析圖………………………………………………….....60
圖三十五、 以原核系統表現PCV2 ORF1、ORF2及ORF3的重組蛋白…….....60

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