辨識與重組B型肝炎大型表面抗原互相影響之細胞蛋白

Abstract

人類B型肝炎病毒(HBV)為世界性的流行病原，在亞洲地區尤其嚴重。它會在人體引發肝臟疾病，而慢性肝炎的病人後期可能轉變為肝硬化、肝腫瘤等。HBV早期感染過程的研究十分困難，因缺乏良好的感染細胞株或小動物模式，直到目前，HBV的細胞受器仍屬未知。HBV外套膜上具有三種表面抗原，分別為大型(large)、中型(middle)、小型(small)蛋白，其中大型表面抗原具有可能的受器結合位置。 本文實驗中，使用兩階段親合性標籤純化(TAP)系統來純化大量的大型表面抗原以及與大型表面抗原結合的蛋白質聚合體，希望從與大型表面抗原結合的細胞蛋白質中發現可能的HBV細胞受器。首先，使用動物細胞株來大量表現含有親合性標籤的重組大型表面抗原，並利用TAP系統純化出大量的重組蛋白。再將重組蛋白與人類原始肝細胞的細胞膜蛋白進行結合實驗，人類原始肝細胞確定可感染HBV並含有HBV細胞受器。之後再次進行TAP純化實驗，純化出與重組大型表面抗原結合的蛋白質聚合體，並利用質譜儀分析，辨認這些結合的蛋白質。 經過多次的改善純化方式與結合實驗，找到了一些會與重組大型表面抗原結合的蛋白質，但並未發現可能的HBV細胞受器，找到的結合蛋白可能與大型表面抗原在細胞中的轉譯後修飾相關。

Human hepatitis B virus (HBV) is endemic in many places in the world, especially in Asian. It may cause serious liver diseases, like liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV cellular receptor is not yet known. Lacking of proper susceptible culture system and animal model makes it difficult to study the early process of virus infection. There are three glycoproteins in HBV surface antigens: large, middle, and small. The large protein is thought contain the cellular receptor binding site. In my study, I use the mammalian tandem affinity purification (TAP) system to purify the recombinant large HBV surface antigen and the interaction complex, to find the recombinant large HBsAg interaction protein. There might be receptor candidates in these interaction proteins. First, purify tagged large protein expressed in mammalian cells. Then, incubate the purified recombinant proteins with human primary hepatocyte membrane proteins, which contain the cellular receptor of HBV. Use TAP system again to purify the interaction protein complexes of large protein. Utilize mass spectrometer to identify these interaction proteins. After several experimental improvements in purification and binding assay, I find some cellular proteins interact with recombinant large protein. But it’s not found any receptor candidate in these interaction proteins. The found interaction proteins are probably related with large protein post-translational modification.