以桿狀病毒表現系統表現蜜蜂囊狀幼蟲病毒之結構蛋白

Using Baculovirus Expression System to Express Structure Protein Of Sacbrood Virus

蔡立宸(Tsai-Li Chen)

指導教授 ： 吳宗遠

中原大學/理學院/生物科技研究所/碩士(2022年)

https://doi.org/10.6840/cycu202200140

摘要

蜜蜂是最重要的經濟性昆蟲，於台灣，自2015年起陸續有蜂農通報在養蜂場和田野間發現大量蜜蜂幼蟲死亡的現象，疑似為蜜蜂感染了蜜蜂囊狀病毒(Sacbrood virus ,SBV)，所引起的蜜蜂囊狀疾病(Sacbrood disease)。SBV主要感染蜜蜂的幼蟲，當蜂群遭受感染後，於蜂巢的外觀可觀察到封蓋的破損，在破損的封蓋當中能觀察到死亡之幼蟲。受SBV感染而死的幼蟲體色為乳白色，體節消失且體表呈現囊狀，蜜蜂囊狀疾病因此而命名。在本研究中，將利用桿狀病毒表現系統，製備同時表現SBV的三個結構蛋白VP1、VP2、VP3之重組病毒，再藉由此重組病毒感染昆蟲細胞，獲得重組蛋白。利用親和性管柱(Anti-HA、Anti-Flag、鎳離子管柱)分析三個結構蛋白之間的關聯性，最後，以穿透式電子顯微鏡驗證有無SBV之類病毒顆粒的存在。由於目前尚無適當的蜜蜂細胞株可供 SBV 病毒感染放大來進行分子生物學的研究，因此本研究若能順利進行，除是第一個製備出 SBV 病毒類病毒粒子的研究外，亦能提供良好的抗原，作為SBV病毒抗體的製備，也在桿狀病毒的應用上，擴展到蜂類病毒的研究，有其學術的重要影響力。

關鍵字

桿狀病毒表現系統 ； 昆蟲細胞 ； 蜜蜂囊狀幼蟲病毒 ； 純化 ； 類病毒顆粒

並列摘要

Bees are the most important economic insects. Since 2015, beekeepers have reported that a large number of bee larvae have died in bee farms and fields. It is suspected that bees have been infected with Sacbrood virus (SBV) disease. SBV mainly infects the larvae of bees. When the colony is infected, the damage of the cover can be observed on the appearance of the hive, and the dead larvae can be observed in the broken cover. The dead larvae infected with SBV are milky white in color, the somites disappear and the body surface is cystic, hence the name of honeybee cystic disease. In this study, a baculovirus expression system will be used to prepare a recombinant virus that simultaneously expresses the three structural proteins of SBV, VP1, VP2, and VP3, and then the recombinant virus will infect insect cells to obtain the recombinant protein. The affinity column (Anti-HA, Anti-Flag, nickel ion column) was used to analyze the correlation between the three structural proteins, and finally, the presence or absence of virus particles such as SBV was verified by transmission electron microscopy. Since there is no suitable honeybee cell line for the amplification of SBV virus infection for molecular biology research, if this research can be carried out smoothly, in addition to being the first research to prepare SBV virus-like particles, it can also provide a good antigen, as the preparation of SBV virus antibody, is also used in the application of baculovirus and extended to the research of bee virus, which has its important academic influence.

並列關鍵字

baculovirus expression system ； insect cell ； sacbrood virus ； purification ； virus like particle

參考文獻

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