在桿狀病毒表現系統中藉由生產牛痘病毒鞘膜蛋白質A26及A27比較共表現及共感染之研究

Compared the co-expression with co-infection methods in Baculovirus Expression Vector System ：Envelope protein A26 and A27 of vaccinia virus

邱璿(Hsuan Chiu)

指導教授 ： 吳宗遠

中原大學/理學院/生物科技研究所/碩士(2016年)

https://doi.org/10.6840/cycu201600867

摘要

生產蛋白質複合物有研究的必要性及實用性，本實驗以牛痘病毒 (Vaccinia virus) 套膜蛋白上的 A26 蛋白質以及 A27 蛋白質複合物 (簡稱為A26-A27蛋白複合物) 之生成為研究模式，比較分別以桿狀病毒表現系統生產A26-A27蛋白複合物時，共感染 (co-infection) 與共表現 (co-expression)這兩種方法之優劣。於共感染的方法上，我們先分別構築可個別表現A26 蛋白質以及 A27 蛋白質的vAc-A26-Rhir-EGFP 和 vAc-A27-Rhir-DsRed2 重組桿狀病毒再各別以兩種不同比例力價之病毒進行共感染實驗；於共表現的方法上，我們以單一多角體蛋白啟動子及其下游的 IRES (Internal ribosome entry site) 序列建構vAc-A26-PnV339-EGFP-Rhir-A27測試其在昆蟲細胞中共表現 A26 蛋白質以及 A27 蛋白質之能力。實驗結果顯示共感染 vAc-A26-Rhir-EGFP 和 vAc-A27-Rhir-DsRed2 重組桿狀病毒，以 4：1 及 3：2 的M.O.I.比例可生產較大量的A26-A27 蛋白質複合物；然以 vAc-A26-PnV339-EGFP-Rhir-A27 單一株病毒感染細胞相較共感染的方式於病毒的製備與感染操作上較為方便，且A26-A27蛋白複合物表現結果較穩定。於能穩定製備A26-A27蛋白複合物後，我們也以質譜儀進行A26-A27 蛋白質複合物的組成分析，結果顯示在 SDS-PAGE 中以 70 kDa 形式存在的蛋白質複合物中為 A26 單體與 A27 二聚體組成， 以 90 kDa 形式存在的蛋白質複合物中為 A26 單體與 A27 三聚體組成。本研究提供一大量表現 A26-A27 蛋白複合物的方法將可進行純化並進行晶體繞射實驗進行結構分析。

關鍵字

桿狀病毒表現系統 ； 共表現 ； 共感染 ； A26 蛋白質 ； A27 蛋白質

並列摘要

The production of protein complexes are a current challenge of biotechnology. Two types of expression methodology via baculovirus-insect cells based expression system were employed to produce vaccinia envelope protein complex A26 and A27, which play an important role in vaccinia virus infection. The first method used single baculovirus vector which contain one promotor and downstream internal ribosome entry site (IRES) to co-expression A26 and A27 protein in insect cells. The second one used two baculovirus vectors contain A26 and A27 gene, respectively, to co-infect insect cells to co-expression A26 and A27 protein. The results showed that co-infection recombinant baculovirus, vAc-A26-Rhir-EGFP and vAc-A27-Rhir-DsRed2, can produce much more A26 and A27 protein in the ratio 4:1 and 3:2. However, In co-expression, infecting cell by single virus vAc-A26-PnV339-EGFP-Rhir-A27 is more convenient and the result is more stable and repoducible. This study also used a mass spectrograph to analyze A26 and A27 protein complex. The result showed that 70kDa protein complex in SDS-PAGE is consist of A26 monomer and A27 dimer And the 90kDa protein complex is consist of A26 monomer and A27 trimer. We can use the coexpression method developed in this study to prepare the A26 and A27 protein complex for further analyze by X-ray diffraction in the future.

並列關鍵字

A26 and A27 protein complex ； Baculovirus expression system ； co-expression ； co-infection ； vaccinia virus

參考文獻

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