臺灣博碩士論文加值系統

傳染性華氏囊病病毒（infectious bursal disease virus; IBDV）為造成三至六週齡雞B淋巴芽細胞壞死之嚴重免疫抑制傳染病。IBDV之主要結構蛋白為VP2及VP3，其中VP2具有誘導中和抗體的抗原表面決定位。為生產供目前應用之較具免疫保護力和診斷意義的IBDV-VP2次單位蛋白，以反轉錄-聚合酶連鎖反應增幅具良好交叉免疫保護力之一新台灣野外IBDV分離株（V263/TW02）之VP2全長約1.5 Kbs基因，選殖後定序，比對其基因親源關係後確定其為已發生點變異之超強毒IBDV（vvIBDV）；以V263/TW02 之VP2基因嵌入桿狀病毒表現系統載體可表達出分子量分別為48和21 kDa之A3和C2次單位蛋白，以該IBDV-VP2-C2製成次單位疫苗以與傳統方法製作之同源病毒不活化IBDV疫苗免疫無特定病原雞比較其免疫保護力，結果VP2-C2次單位疫苗之免疫保護力略遜於傳統不活化疫苗，但卻可激發高的酵素連結免疫吸附試驗抗體反應力價。V263/TW02-VP2-C2次單位蛋白或可供作為IBDV之血清學診斷試驗用之抗原，V263/TW02-VP2-A3次單位蛋白之免疫原性尚待進一步試驗探討。

Abstract
Infectious bursal disease virus (IBDV) is a immunosuppressive infectious disease in causing necrotic of B-lymphoblast in chickens at 3 to 6 weeks of age. Viral protein (VP) 2 and 3 are the mainly important 2 structural proteins of IBDV. The VP2 of IBDV contains the antigenic epitopes in inducing the neutralizing antibody. The whole length of VP2 gene, approximately 1.5 Kb nucleotides, of a recently local isolate (V263/TW02) IBDV being verified with good cross protectivity against several local isolates was amplified with reverse transcriptase-polymerase chain reaction , sequenced and phylogenic analysed of the nucleotide as a very virulent IBDV (vvIBDV) with some point mutation. The gene was then cloned in the vector of the baculovirus expression system for producing the most protective VP2 subunit protein as antigen for IBDV V263/TW02-VP2 subunit vaccine preparation. A3（48 kDa）and C2（21 kDa）2 subunit proteins was found expressied in the system. The V263/TW02-VP2-C2 subunit vaccine was then used to immunized the specific-pathogen-free chickens for comparison of the protectivity with the conventional inactivated V263/TW02 IBDV vaccine against the homologous IBDV challenge. The V263/TW02-VP2 subunit vaccine was found inducing a much higher titer of enzyme-linked immunosorbent assay antibody response but less protective than the inactived V263/TW02 vaccine did. The V263/TW02-VP2-C2 subunit protein might also be good for preparing serological diagnostic kits. The immunogenicity of V263/TW02-VP2-A3 subunit protein need to be further evaluated.

中文摘要………………………………………………………………….. Ⅰ
英文摘要………………………………………………………………….. Ⅲ
中文、英文對照縮寫…….………………………………………………. Ⅳ
誌謝……………………………………………………………………….. Ⅵ
目錄……………………………………………………………………….. Ⅶ
圖次索引………………………………………………………………….. Ⅸ
表次索引………………………………………………………………….. ⅩII
第一章 前言…………………………………………………………….. 1
第二章 文獻回顧……………………………………………………….. 4
2. 1 歷史背景……………………………...…………………………... 4
2. 2 傳染性華氐囊病毒之特性……………………...………………... 5
2. 2. 1 病毒結構及理化特性...…………………………..………… 5
2. 2. 2 IBDV之核苷酸及其蛋白質特性……….…………………. 6
2. 2. 3 IBD發生及急性型IBD的流行病學……………………… 8
2. 2. 4 IBDV之分子流行病學….…………………………………. 10
2. 2. 5 IBDV病毒株的抗原性……….……………………………. 11
2. 2. 6 IBDV病毒株的致病性………………………….…………. 12
2. 2. 7 IBDV病毒株的毒力標誌…………………………………... 13
2. 2. 8 IBDV病毒株之減毒及適應於細胞之培養馴化…………... 13
2. 3 IBDV的致病機致和免疫缺陷性………..………………………. 15
2. 4 IBD的診斷和vvIBDV的特性………………………………….. 16
2. 4. 1 IBD的感染症狀……………..……………………………… 16
2. 4. 2 IBD的分子診斷學………….……………………………… 17
2. 5 VvIBD之預防與控制……………………………………………. 19
2. 6 桿狀病毒表現系統………………………………………………. 21
第三章 材料與方法……………………………………………. ………..23
3. 1. IBD VP2基因之分子選殖…. …………………………………… 23
3. 1. 1 試驗用病毒……………………….………………………… 23
3. 1. 2 病毒之增殖………………...……………………………….. 23
3. 1. 3 病毒之純化……………………………………….………… 23
3. 1. 4 病毒RNA之抽取…………………..…………...…………. 24
3. 1. 5 引子之設計…………….…....................................………… 25
3. 1. 6 反轉錄─聚合酶連鎖反應…………….…………………… 25
3. 1. 7 PCR產物之選殖………………………………….………… 26
3. 1. 8 勝任細胞之製備………………………………….………… 28
3. 1. 9 轉換載體之構築………………...…………………………. 28
3. 2 桿狀病毒表現系統……………………………………………….. 29
3. 2. 1 昆蟲細胞培養…………………..…………………………... 29
3. 2. 2. 共同轉染昆蟲細胞產生重組病毒…..……………………... 31
3. 2. 3. 噬菌斑分析…………………………………….…………… 32
3. 2 4. 使用PCR方式分析重組病毒……………………………… 33
3. 2. 5 偵測重組蛋白質表現情形……………………………...….. 35
3. 3 以傳統方法自製IBD不活化疫苗和桿狀病毒表現系統生產次單位疫苗之免疫保護力比較…………………………………….. 37
3. 3. 1 試驗用病毒……………………………………...…….......... 37
3. 3. 2 試驗雞隻…………………………………………………… 37
3. 3. 3. 傳統不活化疫苗之製備……………………………………. 37
3. 3. 4. 次單位疫苗之製備…………………………….….………... 38
3. 3. 5. 抗原測試……………………………………………………. 38
3. 3. 6. 安全試驗……………………………………………………. 39
3. 3. 7. 效力試驗……………………………………………………. 39
第四章 結果……………………………………………………………. 42
第五章 討論…………………………………………………………….. 51
第六章 參考文獻…..………………….………….……………………. 94
附錄………………………………………………………………………. 104
作者簡介………………………………………………………………….. 131
圖次索引
圖次 頁數
Fig. 1 Reverse transcription-polymerase chain reaction amplified the 1508 bps nucleotides of IBDV-V263/ TW02-VP2 isolate. Lane 1: PCR product of 1508 bps nucleotides; lane 2:negative control; lane M: 100bps DNA marker……………………………………. 58
Fig. 2 Identification of the cloned IBDV-V263/ TW02-VP2 gene by using BamH I and Xho I restriction endonuclease to cut off the gene from pGEM-T vector （3.0 kb）and PCR. Lane 1: the 1508 bps nucleotides PCR product amplified from the recombinated pGEM-T plasmids; lane 2: VP2 gene at 1.5kbs and pGEM-T vector（3.0 kbs）; lane M: 100 bps DNA marker………………. 59
Fig. 3 Identification of the cloned IBDV-V263/TW02-VP2 gene by using BamH I and Xho I restriction endonuclease to cut off the gene from pBlueBacHis2C vector（4.9 kbs）. Lane 1 and 3: the 1.5 kbs nucleotides PCR product amplified from the recombinated pBlueBacHis2C plasmids; lane 2 and 4: the VP2 gene at 1.5kbs and the pBlueBacHis2C vector（4.9 kbs）; lane M: 100bps DNA marker……………………………………………... 60
Fig. 4.1 Comparison of the amino acid sequence in VP2 whole length gene of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package…………………… 61
Fig. 4.2 Comparison of the amino acid sequence in VP2 whole length gene of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package…………………… 62
Fig. 4.3 Comparison of the amino acid sequence in VP2 whole length gene of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package…………………… 63
Fig. 4.4 Comparison of the amino acid sequence in VP2 whole length gene of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package…………………… 64
Fig. 4.5 Comparison of the amino acid sequence in VP2 whole length gene of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package…………………… 65
Fig. 5 Phylogenetic relationship（A）and tree （B） based on amino acid sequence at VP2 whole length gene among of 17 infectious bursal disease viruses using the Clustal program of the DNASTAR software package……………………………………. 66
Fig. 6.1 Comparison of the amino acid sequence （206 to 255） in VP2 gene among 57 infectious bursal disease viruses between 2 enzymes, Acc I and Spe I, cutting site is listed. The large boxed in area （212 to 224）included the hydrophilic region is responsible for the neutralizing epitopes………………………… 67
Fig. 6.2 Comparison of the amino acid sequence （256 to 305） in VP2 gene among 57 infectious bursal disease viruses between 2 enzymes, Acc I and Spe I, cutting site is listed…………………... 68
Fig. 6.3 Comparison of the amino acid sequence （306 to 350） in VP2 gene among 57 infectious bursal disease viruses between 2 enzymes, Acc I and Spe I, cutting site is listed. The large boxed in area （314 to 324）included the hydrophilic region is responsible for the neutralizing epitopes. A conserved region for virulence is underlined…………………………………………... 69
Fig. 7 Phylogenetic relationship tree based on amino acid sequence at hvVP2 gene among of 56 infectious bursal disease viruses using the Clustal program of the DNASTAR software package………. 70
Fig. 8 Amino acid hydrophicity of IBDV-V263/TW02-VP2 gene using the Clustal program of the DNASTAR software package.………. 71
Fig. 9 Result of plaque assay the recombinant baculovirus. The 10-4 dilution folds recombinant baculovirus suspension was found with 13 bluish Sf-9 cell plaques; Control: normal Sf-9 cells……. 72
Fig. 10 Identification of the recombinant plasmid and baculovirus DNA by polymerase chain reaction. Lane 1: PCR product of 1821 bps nucleotide; lane 2: PCR product of 1821 bps recombinant plasmid and 839 bps wild type baculovirus nucleotides: lane 3: normal Sf-9 cell; lane M: 100 bps DNA marker……………….... 73
Fig. 11 Immundot blotting assay for 2 cloned of baculovirus expressed V263/ TW02-VP2 protein. 1: baculovirus expressed V263/ TW02-VP2-A3 protein; 2: baculovirus expressed V263/ TW02-VP2-C2 protein. 3: IBD V263/TW02 virus; 4:normal bursal homogenate; 5: normal Sf-9 cells protein………... 74
Fig. 12 Western blotting assay the VP2 protein of infectious bursal disease virus. Lanes 1-2: normal Sf-9 protein; lanes 3-4: baculovirus expressed V263/ TW02-VP2-C2 protein; lanes 5-6: baculovirus expressed V263/ TW02-VP2-A3 protein……………………………………………………………. 75

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