重組桿狀病毒：屈公病毒的靈敏檢測工具和藥物篩選平臺

Recombinant baculovirus: a flexible detection tool and drug screening platform for Chikungunya virus

穆信(Muhammed Muhsin Varikkodan)

指導教授 ： 吳宗遠

中原大學/理學院/化學研究所/博士(2021年)

https://doi.org/10.6840/cycu202100656

摘要

每年，傳染病都會在全世界造成巨大的痛苦和死亡。特別是，像屈公病毒病毒（Chikungunya virus, CHIKV) 利用節肢動物傳播的疾病正重新出現並大流行，已感染了全世界數百萬人。傳播這些病毒的蚊子正在傳播到全球。由於人類棲息地的擴增和氣候變遷，蚊子與人類的接觸變得更加緊密。 尤其CHIKV正在進化，並正在擴大與蚊子的相容性。因此，改進監測或避免節肢動物傳播病毒的方法具有全球迫切性。為了找到新的抗病毒藥物或植物衍生的化合物來抑制CHIKV的方法，抑制病毒感染和複製是很好的切入點。本論文即在建立診斷與治療 CHIKV 感染的平台。首先，我們成功開發並建立了通過 BEVS 平台檢測 CHIKV 感染的系統。我們分析了四種名為5C5、1E3、1B1和4A1的新型單株抗體（mAbs）並確認其抗原為CHIKV的結構蛋白。我們識別 1E3 和 5C5 mAb 為 辨認CHIKV -E1 的單株抗體而1B1 mAbs 則為識別 CHIKV-E2的單株抗體。同時我們進一步將 1B1 mAbs開發為的 CHIKV 診斷試劑。其次，我們開發了以桿狀病毒表現系統為平台的抗屈公病毒病毒藥物篩選平台。CHIKV的結構蛋白通過內部核糖體入口位點（IRES）同時表現綠色螢光蛋白（EGFP）以利於細胞融合抑制檢測。我們通過這種方法確定熊果酸在體外具有抗 CHIKV 化合物的潛力。然後，基於BacMam病毒的基因傳遞系統，成功地在U-2OS細胞中表現屈公病毒的非結構蛋白，並用於介導亞基因啟動子控制綠色螢光蛋白的表現，作為體外CHIKV複製系統的模擬。利用此一篩選系統我們確定了黃梣苷和黃梣苷元可以抑制亞基因啟動子控制綠色螢光蛋白在U-2OS 細胞中的的表現，顯示我們已開發新的對抗 CHIKV 感染的藥物篩選平台。

關鍵字

700

並列摘要

Every year, infectious diseases cause a great deal of misery and death around the world. Particularly, arthropod-borne viruses like Chikungunya virus (CHIKV) are re-emerging and infecting millions of people all over the world. Mosquitoes that transmit these viruses are spreading to new parts of the globe where there are naive communities to act as viral hosts. Mosquitoes have become more similar to humans due to human habitat encroachment and climate change. The CHIKVs are evolving and are expanding their vector compatibility to include more than one species of mosquitoes. As a result of the combination of these factors, there is a critical global need to improve methods to monitor or avoid arthropod-borne viruses. In order to find novel antivirals drugs or plant derived compounds and ways to inhibit these viruses, a deeper understanding of viral infection and replication is needed. This dissertation will outline a number of works that all aim to improve knowledge or tools for diagnosing, preventing, or treating CHIKV infection. First, we successfully developed and established a system for detecting CHIKV infection through BEVS platform. Here, four novel monoclonal antibodies (mAbs) named 5C5, 1E3, 1B1 and 4A1 generated and used to diagnose structural proteins of CHIKV as antigens from serum samples. Among these mAbs, specifically the CHIKV E1 recognizing both 1E3 and 5C5 mAbs has proved sensitivity against CHIKV. The 1B1 mAbs exhibited adequate specificity to recognize CHIKV-E2 from the samples might be useful for the development of new CHIKV diagnostic approaches that help to prevent the spread of CHIKV. Secondly, we developed baculovirus based compounds assay for the screening of anti-CHIKV phytochemicals in Spodoptera frugiperda insect cells (Sf21) and U-2OS cells. The structural protein of CHIKV was expressed with green fluorescent protein (EGFP) by internal ribosome entry site (IRES) thorough cell fusion inhibition assay approach. We identified that ursolic acid has the potential of anti-CHIKV compound in in vitro by using this approach. Then, BacMam virus based gene delivery has been successfully applied to the transient expression of non-structure proteins with subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. This screening system has identified the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in the U-2OS cells, which may provide a path for the development of new human drug therapies against CHIKV infection.

並列關鍵字

44200

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