開發可區分辣椒葉脈斑駁病毒與番椒葉脈斑駁病毒的核酸和血清檢測方法

辣椒葉脈斑駁病毒（chilli veinal mottle virus; ChiVMV）和番椒葉脈斑駁病毒（pepper veinal mottle virus; PVMV）都是茄科番椒屬作物的Potyvirus屬病毒，且在potyviruses中，二者的鞘蛋白相似度最高，因此有高強度抗血清相關性，即利用純化病毒或鞘蛋白製備的多元抗體無法區分此二病毒。比對ChiVMV與PVMV的鞘蛋白基因與3'非轉譯區，以鞘蛋白N端與3'非轉譯區差異最大。因3'非轉譯區長度差異大，以此區域設計專一性引子進行反轉錄聚合酶連鎖反應（reverse transcription-polymerase chain reaction; RT-PCR），利用增幅產物分子量大小不同，可以區分此二病毒。製備專一性抗血清則利用ChiVMV與PVMV之鞘蛋白N端差異大區域，以ChiVMV之鞘蛋白N端39個胺基酸（4.3 kDa），後接綠色螢光蛋白（28.2 kDa），使其成為複合蛋白加大分子量加強免疫反應，再以pET-28b（＋）為表現載體，於Escherichia coli Rosetta產生一32.5 kDa之複合表現蛋白。大量表現此複合蛋白，加以純化後進行白兔免疫注射，獲得之多元抗體，進行enzyme-linked immunosorbent assay（ELISA）或西方漬染法，均可正確檢出感染ChiVMV之材料，而不與PVMV感染的煙草、甜椒和健康植株反應。用在檢測田間樣本時，與純化病毒製備之抗血清比較，可正確區分樣本是否為ChiVMV感染。比起製備單元抗體，利用細菌表現蛋白製備專一性多元抗血清，可不須經過繁複的步驟即可快速且簡便地得到ChiVMV專一性多元抗血清。

關鍵字

辣椒葉脈斑駁病毒；表現蛋白；多元抗體

並列摘要

Pepper crops are commonly infected with two potyviruses, chilli veinal mottle virus (ChiVMV) and pepper veinal mottle virus (PVMV) in Taiwan. ChiVMV and PVMV share the highest sequence similarity of coat protein genes among potyviruses, preventing serological differentiation between the two viruses. However, the nucleotide sequences between the N-terminal region of the coat protein and the 3' non-coding region (3'-NTR) of two viruses are significantly different from each other. Based on 3'-NTR sequence divergence, ChiVMV and PVMV can be molecularly differentiated by designed primers in reverse transcriptase-polymerase chain reaction (RT-PCR). For serological detection, the ChiVMV-specific antiserum was prepared using a fusion protein as an antigen. The antigen consisted of the divergent and most proximal N-terminal 39 amino acids (4.3 kDa) of ChiVMV coat protein as hapten and green fluorescent protein (28.2 kDa) as a carrier. Both heptan and carrier peptides were constructed into bacterial expression vector pET-28b (+) to produce an expressed fusion protein of 32.5 kDa in Escherichia coli Rosetta. The fusion protein was then immunized rabbits at regular intervals, and the harvested antiserum was used for enzyme-linked immunosorbent assay (ELISA) or Western blotting to detect ChiVMV. The antiserum reacted positively and specifically to ChiVMV, but neither to PVMV nor non-inoculated plants. The ChiVAM-specific antiserum could specifically detect ChiVMV from field-collected samples, even in the presence of PVMV. When compared with the preparation of monoclonal antibodies, using the bacterial express fusion proteins containing ChiVMV partial coat protein to prepare polyclonal antisera is quicker, easier, and more economic.

並列關鍵字

Chilli veinal mottle virus (ChiVMV) ； Expression protein ； Polyclonal antibody

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