臺灣博碩士論文加值系統

豬環狀病毒2型(porcine circovirus type 2, PCV2) 屬於環狀病毒科之環狀病毒屬，是豬環狀病毒相關疾病(porcine circovirus associated disease, PCVAD) 的主要病原體。病毒基因第二開放閱讀框(ORF2) 所轉譯的PCV2衣殼蛋白(capsid protein, Cap) 是PCV2唯一的結構蛋白，並具有誘導宿主免疫反應的主要抗原決定位。由於PCVAD具有PCV2和其他病毒共感染的情形，故設計將豬瘟病毒(classical swine fever virus, CSFV) 醣蛋白E2的中和抗原決定位(neutralizing epitope) WH303 插入PCV2 Cap的loop CD以開發具有二價功能的次單位疫苗。此外，Cap蛋白含有誘餌抗原決定位(decoy epitope)，誘導宿主產生大量不具中和性的抗體，促使病毒逃避宿主的免疫反應。為了避免誘餌反應，遂將誘餌抗原決定位內重要的氨基酸殘基進行突變，並進一步分析類病毒顆粒(virus like particles, VLP) 的抗原性和結構。含有CSFV WH303抗原決定位的重組蛋白rCapCE2和兩個具有誘餌抗原決定位修飾的重組蛋白rCapDEm3和rCapDEm4均可在大腸桿菌中成功地表現。經由快速液態層析儀(fast performance liquid chromatography, FPLC) 純化重組蛋白後，以蛋白質電泳和西方墨點法進行重組表現蛋白分析。進一步以穿透式電子顯微鏡觀察證實此三種重組蛋白均可形成與PCV2病毒顆粒相似的VLP。進一步進行小鼠免疫試驗以評估PCV2 VLP的免疫原性，小鼠免疫血清經酵素結合免疫吸附分析法 (ELISA) 測定之結果顯示次三種重組表現蛋白皆可引起小鼠產生抗PCV2 Cap之特異性抗體，但rCapCE2組並無法檢測到對抗CSFV E2的特異性抗體。綜合以上結果顯示，PCV2 Cap蛋白中誘餌抗原決定位的修飾和外源胜肽插入並不會影響VLP的組裝，並且在小鼠中可以產生針對PCV2 Cap蛋白的特異性體液免疫反應。

Porcine circovirus type 2 (PCV2) is a member belongs to the genus Circovirus of the Circoviridae family, and is the main pathogen of porcine circovirus associated disease (PCVAD). The PCV2 capsid protein (Cap) translated by the second open reading frame (ORF2) is a sole PCV2 structural protein, and contains the major antigenic epitope to induce a host immune response. PCVAD has been found to have co-infection with PCV2 and other viruses. Therefore, a neutralizing epitope (WH303) of the classical swine fever virus (CSFV) glycoprotein E2 was designed to insert at the Loop CD of PCV2 Cap to develop a vaccine with bivalent function. In addition, Cap protein also contains a decoy epitope that induces the host to produce antibodies that are not neutralizing, allowing the virus to escape the host's immune response. In order to avoid the decoy effect, we replaced the amino acid residues within decoy epitope with alanine and further analyzed the antigenicity and structure of virus like particles (VLPs). The recombinant protein containing the CSFV WH303 epitope, rCapCE2, and two recombinant proteins with decoy epitope modification, rCapDEm3 and rCapDEm4, were successfully expressed in E. coli. Recombinant proteins were purified by fast performance liquid chromatography (FPLC) and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Observation by a transmission electron microscope (TEM) confirmed that all three recombinant proteins could form VLPs similar to PCV2 virus particles. The immunogenicity of PCV2 VLP were further evaluated in mice. All three recombinant proteins were able to elicit anti-PCV2 Cap-specific antibody in enzyme-linked immunosorbent assay (ELISA), but the rCapCE2 group was unable to produce specific antibodies recognizing the CSFV E2 protein. All the result show that the modification of the decoy epitope and a foreign peptide insertion do not affect the assembly of VLP, and PCV2 VLPs could mount both humoral and cellular responses against PCV2 cap in mice.

摘要 i
Abstract ii
目次 iv
表目次 vi
圖目次 vii
第一章 前言 1
第二章 文獻探討 3
一、 豬環狀病毒的簡介 3
二、 豬環狀病毒第二型之結構及特性 3
三、 豬環狀病毒第二型基因的複製及表現 4
四、 豬環狀病毒第二型之外殼蛋白 4
五、 豬環狀病毒第二型之致病機制 6
六、 豬環狀病毒第二型之診斷技術 7
七、 豬環狀病毒第二型之預防及疫苗發展 8
八、豬瘟病毒結構性醣蛋白E2簡介 9
九、丙氨酸掃描突變技術 9
第三章 材料與方法 11
第一節 PCV2 Cap重組表現質體之構築 11
一、 消除PCV2 Cap基因誘餌抗原決定位 11
二、 以豬瘟病毒CSFV E2 WH303抗原決定位插入PCV2 C端抗原位 11
三、 DNA片段之純化 12
四、 重組表現質體的構築 12
五、 大腸桿菌轉型作用(Transformation) 12
六、 小量質體DNA之萃取 13
第二節 PCV2 Cap 重組蛋白之表現及分析 14
一、 大腸桿菌表現Cap重組蛋白 14
二、 蛋白質電泳分析(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 14
三、 西方墨點法(Western blot) 15
四、 間接式免疫螢光染色(indirect immunofluorescent assay, IFA) 15
第三節 PCV2 重組類病毒顆粒(VLP)之分析 16
一、 利用快速液態層析儀(fast performance liquid chromatography, FPLC)純化Cap重組蛋白 16
二、 穿透式電子顯微鏡(transmission electron microscope, TEM) 16
三、 免疫金粒子標記技術(Immunogold labeling technique) 17
第四節 PCV2重組類病毒顆粒之抗原性分析 17
一、 小鼠免疫試驗 17
二、 血清學分析 17
第四章 結果 19
第一節 豬環狀病毒第二型重組Cap基因片段rCap DEm及rCapE2的構築 19
第二節 PCV2重組蛋白表面抗原分析 19
一、 PCV2 Cap重組蛋白rCapDEm及rCapE2的表現 19
二、 重組表現蛋白rCapDEm及rCapCE2之純化 20
第三節 PCV2 rCap 類病毒顆粒(VLP)之組裝及分析 20
一、 重組蛋白自行組裝形成類病毒顆粒(VLP) 20
二、 免疫金粒子標定分析 21
三、 間接式免疫螢光染色法分析重組表現蛋白進入細胞的功能 21
第四節Cap 重組表現蛋白之免疫原性分析 21
第五章 討論 34
參考文獻 38

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