在台灣慢性B型肝炎病毒感染是造成肝細胞癌最重要的因素。慢性B型肝炎病毒感染的過程中，病毒的表面抗原會因為免疫壓力的篩選而衍生出突變型，稱之為大型B型肝炎病毒表面抗原pre-S突變型。其中一種突變型因為在表面抗原S基因上的pre-S2區域有部分序列缺失，因而稱之為大型B型肝炎病毒表面抗原pre-S2突變型，近來被認為和肝細胞癌的發展相關。因為大型B型肝炎病毒表面抗原pre-S2突變型會引起氧化壓力、造成染色體不穩定性、並且使得肝細胞容易形成結節，這些現象在在顯示它可能參與在癌化的過程當中。為釐清大型B型肝炎病毒表面抗原pre-S2突變型在肝細胞癌化過程所扮演的角色，過去我們實驗室利用酵母菌雙雜交法找出和它有交互作用的蛋白質，其中之一為Jun活性區域結合蛋白1 (JAB1)，兩者的結合能力也另外利用免疫沉澱法確認屬實，在功能上，我們發現兩者間的交互作用會造成細胞質中的JAB1從連結的MIF脫離並且往細胞核聚集，細胞核中累積大量的JAB1進而造成p27的降解增加，p27無法持續抑制CDK2-cyclin E/CDK2-cyclin A複合體，使得RB被磷酸化，啟動下游基因表現，促使細胞週期持續進行進入S期。JAB1的功能已經被證實，但是JAB1引起的種種現象是否是因為它和大型B型肝炎病毒表面抗原pre-S2突變型直接交互作用而造成的，仍須更直接的證據來支持，因此我的研究目標就是要去尋找出大型B型肝炎病毒表面抗原pre-S2突變型和JAB1直接交互作用的區域。首先我們將大型B型肝炎病毒表面抗原pre-S2突變型創造出幾個部份序列缺失的Clone，將其接入具有GAL4 DNA結合區域的載體pGBKT7中，其中五個突變型是以原生型大型B型肝炎病毒表面抗原當做模板，將大型B型肝炎病毒表面抗原pre-S2突變型在pre-S2區域的基因缺失區域的五十四個核苷酸分成更小的三部份，創造出 LHBS wt △125-134，LHBS wt △130-138和LHBS wt △135-143，而 LHBS wt M120I則是模擬中間起始碼位置由ATG突變成ATA，LHBS wt N123Q代表可能被破壞掉的N-醣化區域 (N-glycosylation site)，另外的八個突變型是利用大型B型肝炎病毒表面抗原pre-S2突變型為模型，將其面向細胞質的區域做一序列缺失而創造出LHBS pre-S2 △1-60，LHBS pre-S2 △1-119，LHBS pre-S2 △1-145，LHBS pre-S2 △1-174，LHBS pre-S2 △203-254，LHBS pre-S2 △203-228，LHBS pre-S2 △229-254和LHBS pre-S2 △361-381 。JAB1基因則是接入具有GAL4 活化區域的載體pACT2上，最後送入酵母菌Y187或AH109中進行酵母菌雙雜交篩選。我們的結果顯示大型B型肝炎病毒表面抗原pre-S2突變型失去其胺基酸序列61到119會完全喪失它和JAB1的結合能力。在pre-S2區域的部分序列缺失都還是能和JAB1有交互作用，因此我們認為pre-S2區域上的序列缺失可能改變了整個蛋白的結構組成，使得原先被包埋在內的區域暴露出來，而外露的區域正好能和JAB1有交互作用。我們期望可以透過此研究對B型肝炎病毒有關的致癌機轉有更進一步的了解，並且找到更好的治療方法。

Chronic hepatitis B virus (HBV) infection is the major cause for hepatocellular carcinoma (HCC) in Taiwan. In the late stages of chronic HBV infection, the pre-S mutant large surface antigen (LHBS) emerges naturally. The pre-S2 mutant LHBS, partially deleted in the pre-S2 region of the surface gene, has been shown predisposed to HCC. The pre-S2 mutant LHBS induces oxidative stress, genomic instabilities, and nodular proliferation of hepatocytes, indicating that it regulates the tumorigenic processes. By using yeast two-hybrid and co-immunoprecipitation assays, we found that Jun activation domain-binding protein 1 (Jab1) is directly associated with pre-S2 mutant LHBS. In our previous study, we found that Jab1 interacts with pre-S2 mutant LHBS and results in p27 degradation, Rb phosphorylation, and S phase progression. In this study, we want to map the domains on pre-S2 mutant LHBS to interact with Jab1. We constructed the partially deleted pre-S2 mutant LHBS genes into the yeast two-hybrid vector pGBKT7, which contains the GAL4 DNA-binding domain. And the Jab1 gene was cloned into pACT2, which contains the GAL4-activation domain. The pair-wise interactions were tested between Jab1 and each pre-S2 mutant LHBS clones by yeast two-hybrid assays in Y187 or AH109 cells. Our data have shown that deletion of amino acid sequences 61 to 119 in the pre-S2 mutant LHBS gene lost its interaction activities with Jab1 completely. In addition, three partial deleted LHBS constructs, covered patient’s pre-S2 deletions, did not loss the interaction activities with Jab1. It is suggested that the deletion in pre-S2 region of the LHBS gene may disrupt the intramolecular interactions of LHBS and expose Jab1 interaction domains. Through these studies, we hope the role of JAB1 as regard to the pre-S2 mutant LHBS-induced HCC can be further understood.

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