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標題: Ag473 蛋白質在 Neisseria spp. 差異表現之特性描述
Differential expression of Ag473 in Neisseria spp.
作者: 陳彗軒
Chen, Huey-Shian
Contributors: 林念璁;曾義雄
楊秋英
中興大學
關鍵字: Neisseria meningitidis
奈瑟氏腦膜炎雙球菌
日期: 2009
Issue Date: 2012-08-31 12:47:58 (UTC+8)
Publisher: 分子生物學研究所
摘要: 脂蛋白 Ag473 為本實驗室近來發現具有開發為腦膜癌疫苗潛力之蛋白質,其功能未明。Ag473 蛋白質表現於所有測試之 Nm 菌株,而以單株抗體 4-7-3 進行 ELISA 及西方墨點分析發現並非所有 Ng 菌株均可偵測到 Ag473 蛋白質。進一步以PCR 分析結果顯示在淋病雙球菌 (Neisseria gonorrhoeae, Ng) 中也有 ag473 基因存在,故本研究主要針對影響 Ag473 在 Nm 與 Ng 中差異表現之機制進行探討。
為確定 Nm 及Ng 中 Ag473蛋白質的表現情形,首先利用α-rAg473 抗血清進行西方墨點法分析,發現所有 Ng 實驗菌株均可偵測到 Ag473 蛋白,且其表現量皆低於 Nm,因此推測 Ng 中之啟動子功能缺失。故利用基因選殖技術將 Ng 及 Nm 之 ag473 及其啟動子基因片段選殖於 pGEM-T easy 載體並送入 E. coli ,發現 Ag473 蛋白質會表現,且兩者表現量相似,此結果顯示 Ag473 之表現量與其啟動子無關。此研究亦發現在重組蛋白質 rAg473-Ng 在SDS-PAGE 分析時移動速率比 Ng 原菌表現的 Ag473 蛋白質 (Ag473-Ng) 快,但Ag473-Nm 及 rAg473-Nm 卻無此差異現象,根據此結果推測 Ag473 蛋白質在 Ng 中所進行的後修飾作用 (post-modification)與 Nm 系統不同。此外,以單株抗體 4-7-3 對重組蛋白 rAg473-Ng 及 rAg473-Nm 進行西方墨點法分析發現單株抗體 4-7-3 對 rAg473-Ng 的辨識力較弱。由以上結果可推論 Ag473 在 Ng 表現量較低,且單株抗體 4-7-3 所辨識的抗原決定位 (epitope) 與 Ag473-Nm 相異。
另一方面,由於 Nm 之Ag473 蛋白質表現量高於 Ng,故利用 RT-PCR 觀察 Nm 與 Ng 中 Ag 473 之 RNA 表現量,發現 Ng 之 RNA 表現量高於 Nm,此結果與蛋白質的表現情形不相符。同步分析 RT-PCR 產物及 chromosome DNA 之 PCR 產物的序列發現 Ng 中 Ag473 表現會發生transcriptional slippage 及 slipped-strand mispairing,而 Nm 中僅發生後者。此外,已解序之 Ng 基因庫中顯示有一轉錄方向與 Ag473 相反之 hypothetical protein (NGO1042),其基因序列與 ag473 基因有重疊。故以 Ag473 蛋白質之 5’ 端之專一性引子分別對於 Nm 及 Ng 進行 RT-PCR,發現反股 RNA 僅存在於 Ng 中。故推論此段反股 RNA (anti-sense RNA) 或許具有抑制 Ag473 蛋白質表現的功能。
Ag473, a novel lipoprotein of N. meningitidis (Nm) recently identified in our laboratory, is a promising vaccine candidate for preventiing the development of disease caused by Nm infection. This protein is detectable by ELISA and Western blotting using monoclonal antibody (mAb) 4-7-3 as the primary antibody in all Nm strains tested but only in a few strains of N. gonorrhoeae (Ng), despite the presence of functional coding region on the chromosomes. The aim of this study was to investigate the molecular mechanism(s) involved in the differential expression of Ag473 in Nm and Ng. First, Western blotting with anti-rAg473 antiserum as the primary antibody was performed to examine the expression of Ag473. Ag473 was found to express in all Ng strains tested, although in significantly lower levels than those in Nm. However, comparable protein levels were expressed when DNA fragments containing the upstream and the coding regions of Ag473 from Nm and Ng were cloned in E. coli, indicating that the promoter is not responsible for the lower expression in Ng. Interestingly, the recombinant Ng Ag473 protein (rAg473-Ng) migrated slightly faster than the endogenous Ng Ag473 (Ag473-Ng), while the mobility was the same for both Nm proteins. Furthermore, rAg473-Ng is recognized by mAb4-7-3 but seems to exhibit lower antigenicity than that of rAg473-Nm. Together, these results suggest that Ng expresses lower amount of Ag473 and its epitope for 4-7-3 is slightly different from that in Ag473-Nm. To address the issue concerning the lower level of Ag473 protein in Ng, RT-PCR was performed to determine the levels of ag473 transcripts. Interestingly, more ag473 transcripts were detected in Ng. Sequencing of the RT-PCR products as well as the PCR products amplified from the chromosomes showed that both transcriptional slippage and slipped-strand mispairing may occur in Ng. In contrast, only the latter situation was found to occur in Nm. Furthermore, antisense RNA was detected in Ng but not in Nm suggesting that the existence of anti-sense RNA may be responsible, at least partly if not all, for the low expression of Ag473 in Ng.
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