Molecular Characterization of Tuberose mild mosaic virus and Preparation of Its Antiserum to the Coat Protein Expressed in Bacteria

利用對應Potyvirus屬病毒基因體RNA3′端之簡併式引子對，可將夜來香微嵌紋病毒(Tuberose mild mosaic virus, TMMV)之RNA模版在反轉錄聚合酶連鎖反應下增幅出一個約2kb之核酸片段。此核酸片段經選殖及核苷酸序列分析後，共解得全長含1947個核苷酸(nucleotide, ut)之序列。經分析比對後登證實此序列的確符合Potyvirus屬病毒3′端基因體之典型架構，由5′端起此序列分別含(coat protein, CP)基因及一個136nt之3′端非轉譯區(3′non-coding region, 3′-NCR)及poly A尾端。此序列經解析後只發現含有一個黑心譯架構，其中所含之部份細胞核內含體b蛋白及全長鞘蛋白乃分別由319及279個氨基酸所組成，預估二者間之蛋白酶切位應落於符合potyviruses特質之Q/S之間，且由鞘蛋白N端起第19-21個氨基酸位置具有代表poyvinuses蚜蟲傳播能力之DAG序列。與已登錄於GenBank之potyviruses基因比對，與TMMY新綠最接近病毒其CP及3′-NCR區域中之核苷酸序列相同度分別只有62及39%;CP之氨基酸序列相同度則低於58%此結果確立TMMV爲Potyvirus屬之新種病毒，同時也印證本實驗室過去以血清學方式證明TMMV乃獨立potyviruses的結果之正確性。根據解得之TMMV CP基因序列，本研究設計對應完整CP基因之專一性引子對，將其順利選殖於表現載體pET-30b上，並使其於E.coli宿主內大量表現一含有部份載體氨基酸序列之融合性TMMV鞘蛋白，普以親和性色層分析法回收純化，經免疫注射製成我元抗體。實驗證明此抗本可應用於ELISA,變重免疫擴散反應及免設點漬法，順利偵測感病夜來香葉片及種球識內之TMMV,其偵測效果與傳統利用純化病毒顆粒所製備之抗體相當。

關鍵字

夜來香微嵌紋病毒； 3′端基因區域；基因序列；表現載體；融合性蛋白；抗血清

並列摘要

A 2-kb DNA product was amplified from purified Tuberose mild mosaic virus (TMMV) virions as well as from infected tissues of tuberose by the use of degenerate primers for potyvirus. The PCR product was subsequently cloned and its sequence analyzed. It was found comprised of 1947 nucleotides (nts) corresponding to the 3′-terminal region of potyviruses. The deduced amino acid sequence contained 598 residues encoding part of the 3′-terminal region of NIb gene (319 residues) and the complete sequence of coat protein (CP) gene (279 residues). A 136 nts of non-coding region (NCR1 was found located at the 3′-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 19-2 1 residues from the N-terminus of CP gene. Compared to the known sequences of potyviruses, the percent of nucleotide identities of the CP gene and the NCR were less than 62% and 39%, respectively. Similarly, percent identities of TMMV’s CP amino acid sequence to those of other known potyviruses were all below 58%, confirming our previous finding that TMMV is a new species of Polyvirus. Using directional cloning technology, a 39-kDa fusion protein containing a complete CP sequence of TMMV and a partial sequence encoded by the expression vector plasmid (pET-30b, Novagene) was highly expressed and purified from E. coli cell cultures. The antigenicity of the fusion protein was determined to be indistinguishable from the viral CP. Antiserum prepared against this fusion protein showed comparable reactivities in the serological detection of TMMV with the conventional antibodies against purified virus particles.

並列關鍵字

3'-terminal region ； Antiserum ； Expression vector ； Fusion protein ； Potyvirus ； Sequences ； Tuberose mild mosaic virus

國際替代計量

Molecular Characterization of Tuberose mild mosaic virus and Preparation of Its Antiserum to the Coat Protein Expressed in Bacteria

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