大量B型肝炎病毒顆粒純化方法之發展

Abstract

人類B型肝炎病毒是帶有外套膜的DNA病毒，內有一個二十面體的核心顆粒。在受到病毒感染的肝細胞中，核心顆粒的組裝是在細胞質中發生，組裝的過程中伴隨著包裹前基因體RNA與病毒DNA聚合酶的結合複合體和一些細胞因子像是熱休克蛋白與會磷酸化核心蛋白的磷酸激酶。磷酸激酶的活性在其他種類的肝炎病毒中也有被發現，並有一些研究鑑定出具有此活性之蛋白質。而熱休克蛋白像是Hsp90、Hsp70、p23都有報導說其對於病毒進行protein priming與病毒顆粒組裝是必需的。這些細胞蛋白質似乎在HBV生活史中，都扮演很重要的角色。因此我們很有興趣去找尋在病毒顆粒中，被特定包裹於其中的細胞蛋白質。 為了能取得大量的病毒顆粒，於是我們選擇使用HBV感染者的血清來當做純化病毒的來源。利用不同濃度硫酸銨沉澱與sucrose cushion來去除血清中大量的血清蛋白像是白蛋白(albumin)。再接著使用蔗糖濃度梯度與氯化銫密度梯度，成功的分離出高純度的病毒顆粒，並同時將表面抗原顆粒與鄧氏顆粒分離出來。我們再利用電子顯微鏡和南方墨點法確認病毒顆粒的結構與鄧氏顆粒內的DNA基因體。最後，我們建立了一套能夠從血清中獲得高純度的病毒顆粒純化方法。

The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral nucleocapsid. In an HBV infected cell, assembly of nucleocapsid occurs in the cytosol and is accompanied by packaging of the viral pregenomic RNA bound to the viral polymerase protein together with cellular protein such as chaperons and protein kinases phosphorylating core protein. This kinase activity is also conserved in other members of hepadnaviridae and plays an important role in viral life cycle. Besides, chaperons like hsp90,hsp70 ,p23 are required for protein priming and encapsidation .Therefore , we are interested in finding out specific cellular proteins incorporated into HBV virions. In order to obtain enough quantity of hepatitis B virus, we chose serum from HBV-infected patients as materials for virus purification. Ammonium sulfate precipitation and sucrose cushion were used to remove high abundant serum proteins, like albumins. After sucrose gradient and cesium chloride gradient, Dane particles and empty particles were obtained with high purity. Then we utilized electron microscopy and Southern-blotting to confirm the structure of particles and virus genome inside Dane particles. Finally, we set up a system for virus purification from serum with high purity.