臺灣博碩士論文加值系統

中文摘要
木瓜畸葉嵌紋病毒(Papaya leaf-distortion mosaic virus, PLDMV) 台灣分離株為馬鈴薯Y病毒科 (Potyviridae)，馬鈴薯Y病毒屬 (Potyvirus group) 之一員，目前發現僅能感染木瓜。在無適當的繁殖寄主以供病毒純化的情況下，為快速製備血清，本研究將木瓜畸葉嵌紋病毒鞘蛋白轉譯區 (reading frame) 基因以專一性引子對，利用反轉錄-聚合酵素連鎖反應 RT-PCR 的方法增幅出，經選殖、解序確定後再將其轉殖到細菌表現載體 pET-32a (+) (Novagen) 中。經IPTG 誘導五小時後的細菌，產生大小約 51.8-kDa 的融合蛋白，其 N 端帶有 109 個氨基酸的蛋白 thioredoxin及六個組織胺基酸 (histidine)。利用先前以細菌所表現帶有纖維素結合區 (cellulose binding domain) 的木瓜畸葉嵌紋病毒鞘蛋白之融合蛋白所製備出之抗血清 As48，及組織胺基酸之專一性單元抗體可於西方轉渍法 (western blotting) 中測定此融合蛋白。此融合蛋白大多存在於不溶性部分 (insoluble fraction)，藉由組織胺基酸與金屬如銅或鎳結合之特性，在變性的條件下 (6 M尿素) 以親和性管柱層析法純化出 51.8 kDa 大小的蛋白，經透析作用去掉尿素後用以製備血清。由於大部分的蛋白未能在透析的過程中還原，所製備出的抗血清 (As 59) 於酵素連結抗體法中，對感病植物中加熱變性的病毒鞘蛋白有很高的靈敏度。該血清也能應用在西方轉漬法 (western blotting) 中去偵測感病植物中的木瓜畸葉嵌紋病毒鞘蛋白。另外，利用本實驗室林氏所建構的矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV) 載體表現PLDMV 鞘蛋白基因，以對應轉譯 (in frame) 的方式插入ZYMV載體之協同性蛋白 (HC-Pro protein) N端，藉由 NIa 蛋白裂解酵素之辨識位置，以利 C 端帶有組織胺基酸標定 (histidine tag) 的游離態蛋白之裂解與純化。含有 PLDMV 鞘蛋白基因的重組病毒ZYMVPCP4-6，在矮南瓜上造成的黃化嵌紋 (yellow mosaic) 及脈綠 (vein-banding) 病徵較 ZYMV 所造成的情況輕微許多。此重組體也能感染刺角瓜造成斑駁 (mottling) 及輕微的脈綠病徵以及在奎藜 (Chenopodium quinoa) 上產生單斑。由此而知本重組病毒 ZYMVPCP4-6 能感染原寄主矮南瓜，同時表現出 PLDMV。

Abstract
The Taiwan isolate of Papaya leaf-distortion mosaic virus (PLDMV), a member of the genus Potyvirus of the family Potyviridae, only infects plants of Carica papaya, which was not the proper propagation host for virus purification. For antiserum production, the complete reading frame of the coat protein (CP) gene of Papaya leaf-distortion mosaic virus (PLDMV) was amplified from the total RNA extracted from virus-infected leaves of papaya plants by reverse transcription-polymerase chain reaction (RT-PCR) with the CP-gene specific primers. The amplified DNA fragment was cloned, sequenced, and transferred to the bacteial expression vector pET-32a (+) vector (Novagen) in this investigation. The PLDMV CP was expressed as a 51.8-kDa fusion protein containing the 109 aa thioredoxin protein and a N-terminal histidine tag in the E. coli cells. This fusion protein reacted with PLDMV-specific antiserum As48, previously produced against the bacterial expressed PLDMV CP protein that was fused with a cellulose binding domain, and the histidine tag-specific monoclonal antibody in western blotting. The target protein of PLDMV CP was collected and purified under denaturing conditions, in buffers containing 6 M urea, by Ni2+-NTA affinity chromatography. The denatured target protein was used for antiserum production after dialysis. Most of the fusion protein was not refolded during the process of dialysis. The antiserum As59 produced by immunizing a New Zealand white rabbit with the purified fusion protein reacted with the boiled extracts of PLDMV-infected papaya plants in ELISA test with high sensitivity. It also was able used to detect the CP of PLDMV in the infected leaves by Western blotting. In addition, the reading frame of PLDMV CP was in frame inserted into the Zucchini yellow mosaic virus (ZYMV) vector at the N-terminal region of the HC-Pro protein, with additional six histidine residues to facilitate the purification and a NIa protease cleavage site to process the free form proteins. The recombinant virus ZYMVPCP4-6 carrying the CP of PLDMV induced milder symptoms of yellow mosaic with vein-banding on zucchini squash plants than those induced by the wild type of ZYMV. The recombinant ZYMVPCP4-6 also induced mottling and mild vein-banding symptoms on plants of horn melon and local lesions on plants of Chenopodium quinoa. The expression efficiency of PLDMV CP in the ZYMVPCP4-6 infected squash plants is discussed.

Content
Abstract……………………………………………………..………………….….. 1
中文摘要………………………………………………………………….………... 3
前言………………...…………………………………………………………….… 5
Introduction…………………………………………………………………...…... 11
Materials and Methods
Virus source……………………………………………………………….……. 17
Reverse transcription-polymerase chain reaction (RT-PCR)……..………….… 17
TA cloning and sequence comparison…………………………………….……. 18
Construction of the reading frame of the CP gene of PLDMV in pET32a vector………………………………………………………………………….
18
Expression and analysis of the target protein…….…………………………….. 19
Purification of the target protein …………………………………………….… 21
Antiserum preparation and serological test…………………………………….. 22
Detection of the titer of the antiserum against PLDMV CP in infected plants by indirect-ELISA……………………………………………………………...
22
Analysis of the sensitivity of the antiserum against PLDMV CP in infected plants by indirect-ELISA……………………………………...………….……
23
Expression of PLDMV CP by the ZYMV vector………………………....…… 24
Plant inoculation……………………………………………………… 24
Detection of the ZYMV recombinants in the infected plants by RT-PCR……... 25
Detection of the recombinant ZYMVPCP4-6 in infected plants by Western blotting……………………………………………………………………….....
25
Detection of the accumulation of viral vector expressed coat protein in infected squash plants…………………………………………………………..
26
Results
Construction of the reading frame of CP gene of PLDMV in pET32a vector………….…………………………………………………………………….
27
Expression of the target gene…………………….…………………………….. 27
Purification of the target protein……………...…………………….....……….. 28
Serological reactivity of the antiserum………………………………..………. 28
Analysis of the sensitivity of the antiserum against PLDMV CP in infected papaya plants by indirect-ELISA………………………………………………
29
Construction of PLDMV CP reading frame into the ZYMV vector………….. 30
Infectivity of the ZYMV vector carrying the CP gene of PLDMV…………… 30
Detection of the PLDMV CP expressed by ZYMV vector in infected squash plants by western blotting………………………………………………
31
The accumulation of viral vector expressed coat protein in infected squash plants……………………………………………………………………
32
Discussion………………………………………………………………………..… 33
Literature cited……………………………………………………………………. 38
Table and Figures…………………………………………………………………. 44
Appendix…………………………………………………………………………... 57

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