豆莢螟核多角體病毒 (MaviNPV) 在 NTU-MV 細胞株系之體外增殖及其 MV74 蛋白特徵分析

Abstract

豆莢螟細胞株 (NTU-MV) 係 2005 年從豆莢螟 (Maruca vitrata) (鱗翅目：螟蛾科) 的蛹內组織所建立，分別利用脂酶 (esterase)、乳酸脫氫酶 (lactate dehydrogenase) 和蘋果酸脫氫酶 (malate dehydrogenase) 三種同功異構酶及轉錄區間段 (internal transcribed spacer regions, ITS) 均能顯示出此細胞株有別於其他已建立的細胞株，且此 ITS 區段與豆莢螟幼蟲的 ITS 區段之相似度高達 98%，因而可證明此細胞株確實來自豆莢螟蟲體。豆莢螟細胞株具有多棘形、圓形、紡錘形和逗號形四種不同形態之細胞，其染色體的個數介於 16 到 268 之間。在含有 8% 胎牛血清之 TNM-FH (Gibco, BRL) 培養液於 28℃ 培養下之生長倍增時間 (doubling time) 為 27 小時，豆莢螟細胞株系是豆莢螟核多角體病毒 (M. vitrata nucleopolyhedrovirus，MaviNPV) 的高接受性細胞株系，在感染 MaviNPV 兩週後，胞外體病毒 (extracellular virus) 的效價高達 3.83 × 107-2.51 × 108 TCID50/ml，且平均每個細胞可生產高達 47 到 50 個封埋體 (occlusion bodies)。此高效率的 MaviNPV 體外增殖系統，可供進行 MaviNPV 基因研究 (mv74) 及構築表現載體之能力。MaviNPV 屬桿狀病毒第一群 (group I) 且和加州苜蓿夜蛾核多角體病毒 (AcMNPV) 親緣關係最為接近。MaviNPV 具 126 個開放譯讀區 (open reading frames, ORFs)，其中有 125 個 ORFs 與 AcMNPV 為同源基因 (homologous genes)，只有 MaviNPV 的 mv74 例外。然而 mv74 與其它四種核多角體病毒有同源基因，但其胺基酸相似度皆很低 (21-26%)。mv74 具 675 個核苷酸，可轉譯出 224 個胺基酸；此基因的早期啟動子，為富含 TA 區域下游 (downstream) 的 第 29 個鹼基對有 CAGT motif，有別於傳統的早期啟動子；其轉錄起始位置符合多數早期基因者位於 CAGT 的 A 鹼基上；mv74 RNA 在病毒感染寄主細胞 2 小時即有低量表現，於 72 小時則達最高量；MV74 蛋白質則在病毒感染 48 小時後方能偵測到，亦於 72 小時達最高表現量。利用免疫螢光偵測發現在病毒感染 48 小時後 MV74 只存在細胞質中，當感染至 72 到 96 小時，同時可見於細胞質與細胞核內，且與封埋體蛋白表現位置有重疊。以免疫金穿透式電子顯微鏡可發現 MV74 分佈於細胞質、胞外體病毒四周或封埋體內及病毒形成團，再經免疫金與負染之穿透式電子顯微鏡觀察，僅看到其位於封埋體上，由此可證實 MV74 為封埋體相關之蛋白質，但其確切功能得待進一步將此基因默化或大量表現後方可確定。

A new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata, was established. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 ℃ was 27 h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase, and lactate dehydrogenase. Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviNPV (M. vitrata nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviNPV and produced high yields of polyhedra (47-50 OBs/cell, 4 × 107-5.96 × 107 OBs/ml) and extracellular virus (3.83 × 107-2.51 × 108 TCID50/ml) after 2 weeks of MaviNPV infection. The NTU-MV cell line and its strains are useful tools for studying the molecular biology of MaviNPV. The MaviNPV genome is relatively small (111, 953 bp) and it contains only 126 putative open reading frames (ORFs). Except MaviNPV mv74, 125 homologous genes of MaviNPV were found in Autographa californica multiple nucleopolyhedrovirus (AcMNPV). MaviNPV mv74 homologs were also found in other NPVs, but mv74 only shared 21 to 26% identities with other NPV homologs in aminoacid sequences. MaviNPV mv74 consists of 675 base pairs encoding 224 amino acids. mv74 contains an early promoter motif, which is defferent from the traditional early promoter motif, and characterized by TA-rich region followed by a CAGT motif 29 bp downstream. MaviNPV mv74 RNA expression could be at first detected at 2 h postinfection, and expression levels reached the peack at 72 h postinfection. The MV74 protein was detected initially at 48 h postinfection with the peak at 72 h postinfection. The MV74 presented in both the cytoplasm and nucleus of the infected cells, especially co-localized with the occlusion bodies (OBs). MV74 was found on the surface of the OBs, but not on the extracellular virus or occlusion-derived virus. We conclude that MV74 is an occlusion body (OB)-associated protein.