萃取及純化第二型豬環狀病毒外殼蛋白類病毒顆粒疫苗

Extraction and Purification of Porcine Circovirus Type 2 Capsid Protein as the Virus-like Particle Vaccine

林欣怡(Lin, Hsin Yi)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2016年)

摘要

豬環狀病毒相關疾病(Porcine circovirus associated disease, PCVAD)為病毒感染所引發的豬隻重要疾病，普遍存在於畜養豬隻國家的豬群中。而第二型豬環狀病毒(Porcine circovirus type II, PCV2)是主要具有致病性的病原，當爆發大流行時皆對養殖業造成重大的經濟損失。施打疫苗為最有效之防疫措施，因此我們希望針對此病毒發展高品質且低成本的疫苗，提供豬隻抵抗病毒感染的免疫力。過去我們實驗室已成功利用桿狀病毒/昆蟲細胞表現系統大量生產PCV2外殼蛋白(capsid protein, Cap)做為疫苗抗原，並且經由豬隻的動物實驗證實的確能夠誘發豬隻對第二型豬環狀病毒的免疫力。本研究藉由發展一套簡單並且可使用於大量生產的程序，針對Cap蛋白進行萃取及純化，並於此程序中保有類病毒顆粒(virus-like particle, VLP)的結構。我們於萃取步驟中先嘗試使用多種清潔劑及鹽類溶液作為萃取液，但在後續發現並無法有效萃取Cap蛋白形成之類病毒顆粒。因此我們改以加入物理性破壞方式於萃取程序中，並且改善物理性破壞方式，使此流程可被使用於大量生產。最後我們確定最佳之萃取程序為使用PBS搭配三次冷凍解凍去除雜蛋白質後，收取細胞碎片沉澱物加入鹽類溶液(20 mM sodium phosphate)並以高壓均質機進行均質。均質後之上清液內Cap蛋白純度由Image J軟體分析純度為38%，並且以此方式可萃取出70%以上的Cap蛋白質。萃取出之Cap蛋白質已可在豬隻的免疫實驗中達到明顯的免疫效果。而在後續純化的部分我們採用工業上常使用的陽離子交換樹脂，藉由改變沖提液鹽類濃度及pH值等條件，已確立可將Cap蛋白純度提升至90%以上之純化流程。最重要的是，我們在穿透式電子顯微鏡分析下，證實此萃取及純化程序可保留PCV2類病毒顆粒的構型。

關鍵字

類病毒顆粒 ； 第二型豬環狀病毒 ； 疫苗 ； 桿狀病毒

並列摘要

Porcine circovirus associated disease (PCVAD) is a prevalent porcine disease. Porcine circovirus type II (PCV2) is the major etiologic pathogen and causes tremendous economic loss. Although the capsid (Cap) protein-based subunit vaccines have been commercialized, the high price of vaccination entails the need for effective and inexpensive vaccines. We have produced PCV2 Cap protein as the vaccine antigen using baculovirus/insect cell expression system and proved the antigen could elicit protective immune response against PCV2 infection in swine. Here we aimed to develop a cost-effective process to extract and purify PCV2 Cap-based vaccine. First, we attempted to use various detergents and salt solutions as extraction buffers, which however resulted in poor extraction efficiency. Therefore, we employed physical methods including sonication and high pressure homogenization in the process and elevated the extraction efficiency significantly. The optimal extraction process involved the use of phosphate-buffered saline and 3 cycles of freeze and thaw, followed by extraction of the Cap protein into 20 mM sodium phosphate with high pressure homogenizer. This process enabled the extraction of Cap protein at an efficiency exceeding 70% and a purity of 38%. Immunization of porcine with the extracted Cap proteins were able to elicit significant immune effects. We further developed a purification process based on cation exchange chromatography. By changing the salt concentrations and pH values of binding and eluting solutions, the purity of Cap protein was increased to higher than 90%. The purified Cap protein self-assembled into virus-like particles, as confirmed by electron microscopy. In summary, we developed an efficient and cost-effective process for the extraction and purification of Cap proteins as the potential PCV2 vaccine.

並列關鍵字

virus-like particle ； porcine circovirus type 2 ； vaccine ； baculovirus

參考文獻

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