蝙蝠研究
2017_利用昆蟲桿狀病毒表達系統探討類冠狀病毒顆粒的組裝_蔡修乙
出版年份：2017
研究生：蔡修乙
分類：碩士論文
題目：利用昆蟲桿狀病毒表達系統探討類冠狀病毒顆粒的組裝
Title：Assembly of Coronavirus-like-particles by Baculovirus Expression System
摘要：
蝙蝠已被證明是嚴重急性呼吸道症候群（Sever Acute Respiratory Syndrome-related Coronavirus, SARS-CoV）的自然宿主，蝙蝠中某些類SARS冠狀病毒已被證實是能夠感染人類細胞，因此本計畫將利用昆蟲桿狀病毒表現系統（Baculovirus expression system）建立一套冠狀病毒類病毒顆粒（Coronavirus-like-particle system）的表現平台，目前已從在台灣分離到的高頭蝠冠狀病毒（Scotophilus kuhlii bat Coronavirus 512 /CYCU-S1/TW/2013）的膜蛋白（Membrane protein, M）、套膜蛋白（Envelope protein, E）以及棘蛋白（Spike protein, S）構築至質體（pFastbacDual及pFastbac）之中，並表現於昆蟲細胞（Spodoptera frugiperda 21），再藉由共感染之方法，組裝成一個完整的冠狀病毒類病毒顆粒，並藉由相似物種之豬流行性下痢病毒（Porcine epidemic diarrhea virus, PEDV）專一性血清檢測，結果顯示冠狀病毒類病毒顆粒的產量及反應不如預期，為了提升產量以及檢測之方便性，我們設計了單一之多基因表現載體，同時表現冠狀病毒M、E及S三種不同之結構蛋白，並分別於結構蛋白上連接了Flag、HA及V5之標誌蛋白，以便後續之檢測，最後再藉由蔗糖梯度超高速離心之方法純化表現的冠狀病毒類病毒顆粒，於西方墨點法檢測得知M與E蛋白能夠正常的組裝且形成類病毒顆粒，而S蛋白則是沒有被正常的表現出來。未來將改變冠狀病毒基因之排列順序，以不同之啟動子改變目標基因之啟動時間以及表現量，藉由此方法能夠更加的優化製作冠狀病毒類病毒顆粒之平台，以及解決部分未表達基因之問題，以達到表現完整的冠狀病毒類病毒顆粒之目的。
Abstract：
Bats have been proved to be the natural host of Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV), some of those SARS-liked Coronaviruses among bats are confirmed to have the ability to infect human cells. Thus, this study is planning to use the Baculovirus expression system to create an expression platform of Coronavirus-like-particle system. At present, we have already constructed the Membrane protein, Envelope protein and Spike protein separated from Scotophilus kuhlii bat Coronavirus 512 /CYCU-S1/TW/2013 in Taiwan into pFastbacDual and pFastbac, perform them on Spodoptera frugiperda 21, using coinfection to compose a complete Coronavirus-like particle, then via the specific serum test of Porcine epidemic diarrhea virus(PEDV), the result shows that there is a weak reaction of Coronavirus-like particle, the production of virus-like particle is not what we expected, in order to improve the amount of production and the convenience of the test, we design single vector of multi-gene expression to perform three different coronaviral structural proteins of M, E and S, and connect them of tag proteins Flag, HA and V5 respectively so as to continue the tests afterwards, ultimately using sucrose density gradient ultracentrifugation to purify the Coronavirus-like particles, knowing M and E protein are able to conjugate and form Coronavirus-like particles via Western blot , while S protein did not present normally. In the future, we are planning to alter the gene sequence of Coronavirus by using different promoter to change the time and the amount it performs of the target gene, through this way renovation of the platform which we use to create Coronavirus-like particles could be accomplished, those problems of nonexpressed gene could also be solved, in order to achieve the goal of performing complete Coronavirus-like particles.