- 學位論文
Tat融合蛋白：Tat-HBx、Tat-Streptavidin的表現與功能分析
Functional analysis of Tat fusion proteins：Tat-HBx and Tat-Streptavidin
摘要
隨著近年來的許多研究，HIV-1 Tat蛋白質已被成功地應用於遞送許多各種大小的物質穿過細胞膜、並進入細胞內，包括了蛋白質、DNA、抗體、影像顯影物質（contrast agents）、藥物，乃至於整個藥物的載體：如微脂粒（liposomes）等等。除了單純的遞送之外，這樣的功能也被成功的應用於蛋白質功能的研究上，特別是一些與細胞凋亡（apoptosis）、細胞週期（cell cycle progression）、和細胞分化（differentiation）等相關的蛋白質。 人類B型肝炎病毒X蛋白質（HBx）是個在Hepatitis B virus（HBV）致病機轉中扮演重要角色蛋白質。近來許多的研究發現HBx具有許多的功能，可以調控基因轉錄、活化訊息傳遞、影響細胞凋亡、控制細胞週期，且與HCC（human hepatocellular carcinoma）的形成有關。然而，許多的研究由於表達的系統不同或是表達量的差異，對於HBx的研究結果仍有許多爭議之處，特別是在於其對細胞凋亡的影響，以及其在細胞內分佈位置的部分。 因此，本研究希望能夠借助於Tat的遞送功能，在將Tat與HBx做成融合蛋白後，利用其在細胞之間均勻分佈的特性，控制HBx在細胞內的數量，在較接近真實HBV感染的條件下，觀察其活性及在細胞內的分佈狀況。 首先，我們利用融合蛋白中的His-tag部分進行蛋白質的純化，同時藉由低溫的誘導表達，成功得到足夠濃度的蛋白質。並利用Western blot方法，分析Tat-HBx融合蛋白進入細胞的條件。在這樣的基礎上，進一步以螢光標定及利用共軛焦顯微鏡，觀察到Tat-HBx是呈現點狀的均勻分佈於細胞質以及細胞核內，這部分與先前大多數研究的結果相同，最後則利用一個具有NF-κB促進序列（enhancer sequence）的螢火蟲螢光素（luciferase）質體（plasmid），將其轉染（transfect）至HeLa細胞後，發現對HeLa細胞處理Tat-HBx，可以轉活化（trans-activate）NF-κB，並造成luciferase冷光訊號的加強，顯示我們所送入的Tat-HBx融合蛋白質具有轉活化NF-κB的能力。 另外我們也構築並表達了Tat與鏈黴親合素（streptavidin，SA）的三種融合蛋白：GST-Tat-SA、GST-SA-Tat、GST-Tat-SA-Tat，希望可以利用鏈黴親合素與生物素（biotin）間，所具有的高度親和與專一性的結合能力，將來應用於遞送標記有生物素的物質進入細胞內。 首先我們利用GST tag（glutathione S-transferase tag）成功地進行蛋白質的純化，並以標記有生物素的quantum dots，進行對HeLa細胞的遞送實驗，顯示GST-Tat-SA、GST-SA-Tat、GST-Tat-SA-Tat皆具有攜帶標記生物素物質進入細胞的能力；另外也以修飾有生物素，包覆doxorubicin的liposomes進行細胞毒殺實驗，發現GST-Tat-SA、GST-SA-Tat、GST-Tat-SA-Tat融合蛋白也可以攜帶liposomes這樣的藥物載體進入細胞，顯示這些融合蛋白具有相當高的應用性。
關鍵字
融合蛋白並列摘要
Recently, HIV-1 Tat peptide has been successfully used to overcome the lipophilic barrier of the cellular membranes and deliver large molecules and even small particles inside the cells for their biological functions. These include some proteins, DNA molecules, antibodies, contrast agents, drugs and nanoparticular drug carriers like liposomes. Besides delivering, this approach has also been used for analyzing protein functions, especially proteins that involve in apoptosis, cell cycle progression and cell differentiation. Human hepatitis B virus X（HBx） protein is an important protein involved in the pathogenicity of Hepatitis B virus（HBV）. Many researches show that HBx is a protein with multiple functions, it can manipulate gene expression, activate signal transduction, influence apoptosis and cell cycle progression, and have been related with human hepatocellular carcinoma（HCC）. Although so many researches have been done on HBx, different researches using different expression systems with different expression levels showed many contradictions, especially the influences on apoptosis and the sub-cellular localization of HBx. For this reason, we used the Tat transduction domain to design a Tat-HBx fusion protein. Taking advantage of the almost equal amount distribution between individual cells and controllable concentration in cells, we tried to analyze the Tat-HBx activity and subcellular distribution of Tat-HBx. We used low temperature induction method to express the fusion protein in E. coli, and purify the fusion proteins by the N-terminal His-tag. Then, the conditions for transduction Tat-HBx into cells were analyzed by Western blot. We also used fluorescence-labeled Tat-HBx and a confocal laser scanning microscope, found that the Tat-HBx protein was homogenous distributed both inside the nucleus and cytoplasm, consistent with most other researches. Finally, we used a reporter plasmid that have a luciferase reporter gene controlled by a NF-κB enhancer sequence, found Tat-HBx have the transactivtion activity for NF-κB just like wild type HBx. Beside the Tat-HBx fusion protein, we also constructed three Tat-streptavidin fusion proteins：GST-Tat-SA、GST-SA-Tat and GST-Tat-SA-Tat. Taking advantage of the high affinity and specificity between streptavidin and biotin, we found these fusion proteins can deliver biotinylated quantum dots into HeLa cells. In a cytotoxicity experiment, we also found these fusion proteins could successfully deliver drug carriers — liposomes into the cells. So these fusion proteins might be useful tools for further applications.