可延遲細胞裂解之桿狀病毒的分析及應用

Abstract

桿狀病毒為節肢動物病毒，其宿主包括鱗翅目、膜翅目、雙翅目和十足目，常見寄宿於鱗翅目幼蟲。1983年桿狀病毒首次被使用於表現外源蛋白，此後廣泛應用在疫苗開發、新藥開發等蛋白質生產。而提高桿狀病毒表現系統的產率至今仍是致力研發的重點。本實驗室在過去利用BrdU致變劑對桿狀病毒vABhcmEpL進行隨機突變，在候選突變株當中篩選出感染昆蟲細胞Sf21後造成裂解延遲的桿狀病毒突變株C4，並且發現與母株相較其綠螢光表現較為強烈。本實驗目的便是要對C4桿狀病毒在蛋白質表現與病毒生產方面進行定性。實驗結果中發現，C4可在為期八天的感染實驗中表現相較於母株病毒長久之綠螢光蛋白 (EGFP)，且能夠生產較多的病毒顆粒。進一步透過次世代定C4病毒與母株比對，試圖尋找造成裂解延遲與外源蛋白表現上升之基因。定序結果發現，共有73個位點突變。配合前人利用轉染桿狀病毒cosmid回補C4細胞裂解功能的試驗，發現其中最有可能造成該表徵的突變基因為ORF52，進一步透過建構C4表現野生型ORF52之重組病毒，則發現細胞裂解時程與一般野生型病毒一致。另一方面，我也利用C4表現流感病毒血球凝集素蛋白 (Hemagglutinin)，並與商業化的桿狀病毒 (BaculoGoldTM) 表現系統比較，發現C4比起BaculoGoldTM更能延遲細胞的裂解，且也產生較多的病毒顆粒，也同時表現較多的血球凝集蛋白。

Baculoviruses are arthropodian pathogens, infecting lepidoptera, hymenoptera, diptera, and decapoda hosts. Baculovirus infects most commonly in the larvae of moths. In 1983, baculovirus was first used to express exogenous proteins. From then on, this technique has been widely used in vaccination, drug discovery and recombinant protein production. Increasing protein yields of baculovirus expression vector system (BEVS) has long been an important goal. Previously, our laboratory have used the mutagen, BrdU, to induce random mutation to the vABhcmEpL baculovirus, and identified a clone C4 virus with a delayed cell lysis phenotype after infecting Sf21 insect cell. C4 virus also expresses higher levels of EGFP than does parental virus vABhcmEpL. The purpose of my experiment is to characterize the C4 virus, focusing on protein expression and virus production. According to current experimental results, I found that C4 could express EGFP for longer time and produce higher virus titer than vABhcmEpL in the eight-day time course experiment. In addition, I utilized NGS to analysis genome of C4 in order to find out the responsive gene(s) to these phenotypes. The NGS result showed that there are 73 mutants different from vABhcmEpL. Comparing the results with cosmid transfection tests done by previous study, I discovered that mutant ORF52 may be responsible for the delay cell lysis phenotype of C4 virus. Therefore, I constructed recombinant baculoviruses based on ORF52 of wild type and compared the phenotype with C4. The result show that vC4-ORF52 induced cell lysis earlier than vC4. Therefore, the mutant ORF52 of vC4 is proved to be the gene results in the phenotype of delayed cell lysis. On the other hand, I also took advantage of C4 to better express the hemagglutinin of influenza virus. The results showed that C4 is not only resulted delayed cell lysis but also produced more virus particles and engineered HA protein than the BEVS derived from commercial vector system of BaculoGold.