以桿狀病毒表現系統生產重組蛋白質疫苗之產程開發與最適化

Recombinant Protein Vaccine Production using the Baculovirus Expression System: Development and Optimization of Production Process

高宜均(Yi-Chun Kao)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2013年)

摘要

桿狀病毒/昆蟲細胞表現系統發展至今已廣泛應用於外源基因表現及重組蛋白質的生產。本研究透過上游基因工程及下游反應器工程兩個不同層面，針對第二型豬環狀病毒外殼蛋白及腸病毒71型類病毒顆粒疫苗兩個主題，來提升桿狀病毒表現系統應用於疫苗生產的潛力。在第一部分的研究中我們延續實驗室先前利用潮汐式填充床生物反應器BelloCell® (0.5 L)來進行腸病毒71型(EV71)類病毒顆粒疫苗的大量生產。我們針對BelloCell®反應器中的生產條件，包括培養基、病毒感染時機、潮汐停滯時間等進行最適化，提升類病毒顆粒的產量。完成生產條件的最適化後，我們進一步將產程放大規模到10 L的TideCell®反應器中，並大幅提升類病毒顆粒產量至約250-300 mg/L。在第二部份的研究中，我們利用桿狀病毒表現系統生產第二型豬環狀病毒的外殼蛋白，透過修改重組桿狀病毒建構的基因工程方法來改變產物在細胞中分佈的位置，由原先侷限在細胞核中變為能夠停留在細胞質中，提升外殼蛋白的產量至170 mg/L，並成功以簡單的化學萃取方式萃取出胞內產物，以利後續純化，使其更具有成為商業化疫苗的潛力。

關鍵字

桿狀病毒 ； 蛋白質疫苗

並列摘要

The baculovirus/insect cell expression system is widely used for the production of recombinant proteins. To exploit the potential of baculovirus system for vaccine production, this study aimed to develop efficient strategies for the production of porcine circovirus type II (PCV2) capsid protein and enterovirus 71 (EV71) virus-like particles (VLP) by genetic and bioreactor engineering approaches. In the first part of this study, we employed baculovirus as a tool for mass production of EV71 VLP using BelloCell® bioreactor, which is a 500 ml, disposable packed-bed bioreactor. We optimized the production conditions in the process, including culture medium, time of infection (TOI) and bioreactor parameters to improve the volumetric yield of EV71 VLP. After determining the process parameters in BelloCell® system, we further scaled the production process up using a 10 liter TideCell® bioreactor for pilot production of EV71 VLP. With the process optimization, the VLP yield was tremendously enhanced to 250-300 mg/L. In the second part of this study, we employed baculovirus system for the production of the major antigen (Cap protein) of PCV2. We designed multiple recombinant baculoviruses in order to change the location of Cap. After modification, the Cap protein resided in the cytoplasm instead of being retained in the nucleus, which led to the enhanced yield to 170 mg/L. We also successfully developed a simple method to extract intracellular product, making it simpler for downstream bioseparation. These works collectively contributed to the optimized production of the PCV2 and EV71 vaccines.

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