臺灣博碩士論文加值系統

豬生殖與呼吸症候群病毒(porcine reproductive and respiratory syndrome virus, PRRSV)是近年來引起豬隻普遍性感染的新病毒，屬於Arteriviridae，基因體全長15.1 kb且包含8個open reading frames(ORFs)。從已知的文獻中可知，ORF5基因轉譯之蛋白是PRRSV結構蛋白中，與中和抗體產生最有關的蛋白。所以本篇論文的主要目的，是針對台灣分離株FI，利用體外轉譯蛋白當抗原進行免疫及直接DNA免疫兩種方式，探討其ORF5基因轉譯之蛋白是否具有誘發宿主產生中和抗體的能力。抽取台灣分離株FI之病毒核酸，利用反轉錄聚合鏈反應(RT-PCR)，增幅出621 bp之ORF5基因片段，再將此ORF5基因片段植入pMT/Bip/V5-HisA表現載體中，經篩選後，選擇嵌有ORF5基因之表現質體(pMT-ORF5)，藉由lipofectin reagent，將此表現質體轉染至Schneider 2細胞內，進行真核系統之蛋白質表現，經硫酸銅誘發蛋白質表現後，利用抗ORF5基因轉譯蛋白之單源抗體，以西方轉漬法(Western blotting)偵測到有28 kDa之蛋白質表現產物，將此蛋白質表現產物免疫BALB/c鼷鼠，經補強注射後，採血進行中和抗體試驗。結果顯示，PRRSV之ORF5基因利用昆蟲表現系統進行蛋白質表現，雖然能成功地產生28 kDa之蛋白質產物，但此蛋白質產物僅能誘發低量的中和抗體產生。此外，進行DNA免疫時，將抽取之病毒核酸，利用重新設計的引子，進行反轉錄聚合鏈反應，再將此PCR產物植入pKAN3224載體中，經篩選後，選擇嵌有ORF5基因之表現質體(pKAN-ORF5)，直接以肌肉注射之方式免疫BALB/c鼷鼠，經注射後採血，進行中和抗體試驗。試驗結果可見，所有實驗組之BALB/c鼷鼠皆有抗PRRSV之中和抗體產生。因此，由實驗結果證明，PRRSV之ORF5基因轉譯之蛋白，確實具有誘發宿主產生中和抗體的能力，且以DNA免疫的方式較以體外轉譯蛋白進行免疫方式更能有效地誘發中和抗體反應的產生。

Porcine reproductive and respiratory syndrome virus represents the causative agent of a new porcine disease which spread in form of an epidemic all over the world in the past years. It belongs to the newly proposed virus family Arterividae. The genome of PRRSV is about 15.1 kb in length and contains eight open reading frames. In the previous studies demonstrated that the ORF5-encoded glycoprotein of PRRSV was associated with neutralizing epitopes. The purpose of the present study is to investigate the ability of ORF5-encoded glycoprotein of PRRSV to elict the neutralizing antibodies in host by means of expressed protein immunization and DNA immunization. The viral RNA was extracted from FI-infected MARC-145 and the ORF5-encoding region was amplified by RT-PCR. The PCR product was inserted into the pMT/Bip/V5-HisA vector at the KpnI/EcoRI site. The Schneider 2 cell was transfected with pMT-ORF5 plasmid by lipofectin reagent. The 28 kDa recombinant protein was expressed after cupper sulfate inducing and detected with anti-ORF5 encoded protein monoclonal antibody by Western blotting. Then, the intraperitoneal injection of recombinant protein into BALB/c mice was performed. Serum samples were collected and checked for PRRSV-specific neutralizing antibodies by virus neutralization test. The results show that recombinant protein expressed in the insect expression system could only induce low-level neutralizing antibodies. In addition, another approach to evaluate the ability of ORF5-encoded protein of PRRSV to induce the neutralizing antibodies in host is direct DNA immunization. The ORF5 gene fragment was amplified by RT-PCR using the redesigned primers and cloned into the pKAN3224 vector. BALB/c mice received intramuscular injection of pKAN-ORF5 plasmid bilaterally into the anterior tibial muscle. After that, serum samples was collected and tested for PRRSV-specific neutralizing antibodies by virus neutralization test. The results indicate that PRRSV-specific neutralizing antibodies were detected in serum of all DNA-immuned BALB/c mice. Consequently, the data obtained suggest that ORF5-encoded protein can indeed elict neutralizing antibodies in the BALB/c mice. Furthermore, the ability to induce neutralizing antibodies by DNA immunization significantly exceeds that induce by expressed protein immunization.

目次
目次………………………………………………………………….I
表次…………………………………………………………………IV
圖次…………………………………………………………………V
摘要………………………………………………………………….1
英文摘要…………………………………………………………….3
第一章 緒言…………………………………………………………5
第二章 文獻探討……………………………………………………7
一、歷史背景……………………………………………………7
二、臨床症狀與病理學…………………………………………8
三、豬生殖與呼吸症候群病毒之結構及生化特性……………9
四、病毒基因與結構蛋白之研究……………………………..11
五、病毒抗原性之研究………………………………………..14
六、診斷………………………………………………………..17
七、DNA疫苗的發展………………………………………..19
第三章 材料與實驗方法…………………………………………..21
第一節 材料…………………………………………………….21
第二節 實驗方法……………………………………………….30
一、豬生殖與呼吸症候群病毒ORF5基因之蛋白質表現…...30
1.病毒的增殖……………………………………………..30
2.病毒核酸的萃取………………………………………..31
3.引子之設計……………………………………………..31
4.反轉錄聚合鏈反應………………………………..32
5.接合反應………………………………………………..32
6.轉形作用………………………………………………..33
7.質體的篩選與製備……………………………………..33
8.核酸定序………………………………………………..35
9.轉染作用………………………………………………..37
10.西方轉漬法……………………………………………38
11.實驗動物之免疫………………………………………39
12.中和抗體試驗…………………………………………39
二、DNA免疫………………………………………………….40
1.DNA質體之製備……………………………………….40
2.間接螢光抗體染色試驗………………………………..40
3.實驗動物之免疫………………………………………..41
4.中和抗體試驗…………………………………………..41
第四章 結果………………………………………………………..42
一、豬生殖與呼吸症候群病毒ORF5基因之蛋白質表現…..42
1.ORF5基因的選殖與表現載體的構築………………………42
2.選殖基因的定序反應………………………………………..43
3.轉染作用之確認……………………………………………..43
4.重組蛋白之蛋白質表現與西方轉漬試驗的確認…………..44
5.血清中和試驗………………………………………………..44
二、DNA免疫………………………………………………….45
1.ORF5基因的選殖與表現載體的構築………………………45
2.選殖基因的定序反應………………………………………..45
3.間接螢光抗體染色試驗……………………………………..46
4.血清中和試驗………………………………………………..46
第五章 討論………………………………………………………..62
一、豬生殖與呼吸症候群病毒ORF5基因之蛋白質表現…..63
二、DNA免疫………………………………………………….67
三、結論與展望………………………………………………..72
參考文獻…………………………………………………………..73
表次
表一、以重組蛋白Bip-gp5,PRRSV活毒及DES expression buffer免疫BALB/c鼷鼠後進行中和抗體試驗之結果………….48
表二、以pKAN-ORF5質體,PRRSV活毒,pKAN3224載體及PBS免疫BALB/c鼷鼠後進行中和抗體試驗之結果………….49
圖次
圖一、PRRSV基因體結構及轉錄方式………………………….12
圖二、PRRSV核酸利用引子P5F/P5R進行RT-PCR之電泳結果…………………………………………………………50
圖三、PMT/Bip/V5-HisA載體之圖譜……………………………51
圖四、轉殖株經快速篩選後，進行電泳之結果………………….52
圖五、轉殖株經小量質體製備法萃取質體DNA，以KpnI及EcoRI切割之電泳結果 …………………………………………..53
圖六、轉染作用後，抽取細胞DNA，利用引子P5F/P5R進行PCR之電泳結果…………………………………………….54
圖七、轉染作用後，抽取細胞DNA，以KpnI及EcoRI切割之電泳結果………………………………………………………….55
圖八、經轉染作用並以硫酸銅誘發蛋白質表現後，進行西方轉漬法偵測之結果……………………………………………...56
圖九、PRRSV核酸利用引子P’5F/P’5R進行RT-PCR之電泳結果……………………………………………………………57
圖十、轉殖株經快速篩選後，進行電泳之結果………………..58
圖十一、轉殖株經小量質體製備法萃取質體DNA，以EcoRI及BamHI切割電泳結果…………………………………..59
圖十二、pKAN-ORF5表現質體轉染至Vero細胞後，以間接螢光抗體染色法檢測之結果………………………………..60
圖十三、以pKAN-ORF5表現質體及PRRSV活毒免疫BALB/c鼷鼠後，進行中和抗體試驗之結果………………………..61

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