臺灣博碩士論文加值系統

豬繁殖與呼吸道症候群(porcine reproductive and respiratory syndrome, PRRS)，是目前世界上廣泛的豬隻傳染疾病，在1987年首次被美國報導出來，其病原體PRRS病毒(PRRSV) 為一種具有外套膜的正股RNA病毒，對於懷孕母豬造成生殖道上的缺陷，以及透過感染豬隻鼻黏膜的上皮細胞以及肺部的巨噬細胞、單核球細胞，使得豬隻的免疫系統受到破壞，造成豬隻免疫力的下降，易於遭受二次感染，而導致死亡。由於現今缺乏良好的疫苗，豬繁殖與呼吸道症候群對於我國的養豬事業造成嚴重的衝擊。因此，本研究希望能直接針對台灣本土的豬繁殖與呼吸道症候群病毒發展出更有效的疫苗。
由先前的研究中指出，PRRSV的GP4、GP5、N基因為動物體免疫系統產生中和抗體的目標部位，以及M基因則是誘發細胞性免疫反應的重要抗原位。所以，我們首先將台灣本土分離株PRRSV-KB3，利用反轉錄聚合脢連鎖反應的方法將病毒的GP4、5、6、7四段基因選殖出來，再利用實驗室中已建立的假性狂犬病病毒(pseudorabies virus, PRV)載體，經由連續的藥物篩選，可將選殖的基因插入假性狂犬病病毒gD基因的位置。最後，可產生帶有PRRSV基因的重組假性狂犬病病毒，命名為PrV-CT4、5、6、7。由於重組病毒缺損gD基因，因此只能造成一次的感染，無法再製造出具感染力的病毒後代，提高了重組病毒疫苗的安全性。在小鼠的實驗中，我們發現此重組病毒疫苗的確能引起對抗PRRSV的抗體免疫反應。在未來，希望此疫苗對於國內豬隻的傳染病防治能有所助益。

Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory illness in young pigs, is a worldwide porcine disease. It was first reported in 1987 in USA and described as mystery swine disease. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped positive-stranded RNA virus. It can infect pigs through nasal epithelial, pulmonary macrophages and monocytes, and cause immunosuppression. Thus, PRRSV is considered to be a major problem to the pork industry. Several PRRS vaccines are available; however, these vaccines confer protection against clinical disease, but do not prevent viral infection. Furthermore, since PRRSV is genetically and antigenically heterogeneous, it is worthwhile to develop new PRRS vaccines based on Taiwan local strains. The aim of this study was to develop pseudorabies virus (PrV) vectors carrying PRRSV genes as vaccines. Therefore, we cloned the ORFs 4, 5, 6 and 7 of PRRSV that encode virion-associated protein (GP4), envelope (GP5), membrane protein (M) and nucleocapsid protein (N), respectively, by RT-PCR. GP4, GP5 and N proteins can produce neutralizing antibodies directed against the virus. In addition, M protein can induce cell-mediated immunity. Therefore, these genes are good targets and important to induce protective immune responses. Using an acycolvir selection system to generate recombinant pseudorabies virus (PrV), the cloned PRRSV genes were inserted in place of the HSV-TK gene of a gE/gD-negative PrV, resulting in the generation of PrV-CT4, 5, 6 and 7. By serial acyclovir selection, the parent virus was killed and recombinant PrV vectors were produced. Because the recombinant PrV vectors lack gD gene, they could not generate infectious progeny, and thus increase the safety. After immunization of the PrV-CT4, 5 and 7, mice could induce anti-PRRSV neutralizing antibody against viral infection. In conclusion, the recombinant PrV is an adequate vector to express and present antigens of PRRSV. These recombinant viruses may be used as vaccines against PRRSV in the future.

Contents
Abstract…………………………………………………………………………..I
Chinese Abstract…………………………………………………………………II
Acknowledgement……………………………………………………………….III
Contents……….…………………………………………………………………IV
Figure contents…………………………….……………………………………..VI
Table contents…………………………………………………………………….VII
Appendix…………………………………………………………………………VIII
Abbreviation………………………….…………………………………………..IX
1. Introduction
1.1 Porcine reproductive and respiratory syndrome virus …...…………….1
1.2 Development of PRRS vaccine……………...…………………………2
1.3 Development of Pseudorabies virus as a vaccine vector……...………..2
1.4 The strategy to produce PRRS vaccines and induction of immunity against PRRSV………………………………………………………… 5
2. Materials and methods
2.1 Cells and viruses…………………………………………………....…..6
2.2 Cloning of the cDNAs of PRRSV-ORF4, 5, 6 and 7...…..…..………...6
2.3 Construction of pTCY/KB3-orf4, 5, 6 and 7 vectors…….…….……….7
2.4 Transient expression of PRRSV proteins………………………………8
2.5 Constructions of pDelta-gD/KB3-orf4, 5, 6 and 7 shuttle vectors..……8
2.6 Productions of gD-/gE-/Tk-/PRRSV genes+ PrV…..…………………...9
2.7 Isolation of Viral DNA………………..…………………….………….9
2.8 PCR detection of PrV-CT4, 5, 6 and 7…………………………..……..10
2.9 Transgene expression in Vero cells infected with PrV-CT……..………11
2.10 Determination of TCID50 of PrV-CT.….……………………………...11
2.11 Immunization of mice with PrV-CT4, 5, 6 and 7….………………….12
2.12 Detection of anti-PRRSV and PrV antibody by ELISA………………12
2.13 Detection of anti-PRRSV neutralizing antibody by neutralizationantibody
(NT) test… …………………………………………………..13
2.14 Detection of anti-PrV neutralizing antibody by NT test………………13
2.15 Lymphocyte proliferative responses to PRRSV……………………….14
3. Results:
3.1 Cloning and sequencing of cDNA of the ORFs 4, 5, 6 and 7 of PRRSV strain
KB3. ……………………………………………………………...15
3.2 Transient expression of the cloned ORF4, 5, 6 and 7 genes by IHC
staining………………….…………………………………………..…..15
3.3 Construction of the pDelta-gD/KB3-orf4, 5, 6 and 7 vectors…………..16
3.4 Detection of recombinant PrV-CT4, 5, 6 and 7 by PCR………………..16
3.5 Transgene expression in Vero cells infected with recombinant PrV……16
3.6 Detection of antibody responses by ELISA…...………………………..17
3.7 Detection of anti-PRRSV antibodies by NT test……………………….17
3.8 Induction of lymphocyte proliferative responses in mice by immunization with recombinant PrV…………………………………...18
4. Discussion……………………………………………………………………..19
References………………………………………………………………………..22
Figures……………………………………………………………………………25
Table……………………………………………………………………………...43
Appendix…………………………………………………………………………46
自述

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