臺灣博碩士論文加值系統

C型肝炎病毒 (HCV) 是目前造成國人罹患肝癌的重要因素之一，在C型肝癌病毒的組成中，有一種非結構性蛋白 (Non-structural protein 5B, NS5B)， 它是RNA dependent RNA polymerase，主要功能是製造HCV的核酸。HCV目前區分成六種不同的基因型，它的分類是根據NS5B的胺基酸序列的差異來作分類的依據。在台灣較為常見的有基因型一與基因型二。Akt/PKB在細胞內能調控許多的功能，包括了細胞的生長、代謝與生存等等，因此，Akt對於細胞來說是非常重要的一種蛋白質。而Akt的主要機制在於能夠磷酸化受質蛋白質，進而調控蛋白質的活性而影響細胞的功能。在對Akt的受質蛋白作胺基酸序列分析後發現，若是蛋白質有RxRxxS/T的這一段特殊的序列時，Akt便會將S/T的位置磷酸化，進而調控蛋白質的活性。在NS5B的胺基酸組成中，基因型一的具有RxRxxS這個Akt受質的特殊序列，因此，NS5B可能是Akt的受質而被磷酸化。為了證明NS5B是否是Akt的受質蛋白，我們實驗室利用了大腸桿菌系統 (E. coli expression system)與昆蟲細胞表達系統 (Baculovirus expression system) 來作NS5B蛋白質的純化與分析。在大腸桿菌系統中，我們利用兩種不同的重組蛋白質體來製造NS5B重組蛋白，它們分別是pGEX-5X-3與pET30b，這兩種不同的質體分別帶有不同的純化標籤以利後續的純化步驟。我們嘗試了許多不同的誘導條件但是只能在西方點墨法的結果裡看到重組蛋白的表現，無法做出大量的重組蛋白。在昆蟲細胞表達系統中，我們成功的製造出重組病毒並且能表達出NS5B重組蛋白。我們利用ÄKTAprimeTM這套蛋白質純化系統，配合離子交換管柱 (Q column) 與肝素鍵結管柱 (Heparin column)來純化由昆蟲細胞所表現的NS5B重組蛋白。得到的重組蛋白質與購買的Akt-1重組蛋白利用γ-32P標定的三磷酸腺苷酸 (ATP) 進行磷酸化反應。結果發現NS5B的確能被Akt所磷酸化。為了更進一步證實不同基因型的NS5B的差異，我們利用免疫沈澱的方式去排除純度與濃度的差異來觀察NS5B的磷酸化。結果顯示NS5B基因型一的確能被Akt所磷酸化。

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. It belongs to the family of Flaviviridae. There are six major genotypes of HCV and the most common in Taiwan is 1b. HCV can generate more than ten proteins. The non-structural protein NS5B, identified as a RNA dependent RNA polymerase (RdRp), plays an important role in HCV replication. We noticed that the NS5B of HCV genotype 1 has the consensus sequence of the Akt/PKB substrate. Akt/PKB is a Ser/Thr-protein kinase which plays important roles in the cell such as cell metabolism, glucose uptake, and cell survival. In this study, we aimed to prove that the NS5B is a substrate of Akt. We constructed the 6xHis and GST tagged NS5B recombinant protein in E. coli expression system. We also used the baculovirus expression system to generate recombinant NS5B with genotype 1b and 2b in insect cells. After generation of recombinant NS5B protein from E. coli system and baculovirus expression system, we used the ÄKTAprimeTM system coupled with Q and heparin column to purify these two kinds of NS5B and the anti-FLAG M2 resin also used to capture these proteins. These purified NS5B proteins were subjected to the Akt kinase assay and immuno-complex Akt kinase assay to determine whether HCV NS5B could be phosphorylated by Akt. We were unable to induce sufficient amount of recombinant NS5B in soluble form from E. coli, though the bacteria could be induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30 ℃ for 4 hours. We succeed in generating recombinant NS5B of both genotype 1b and 2b from baculovirus expression system followed by purification by ÄKTAprimeTM system coupled with columns. The purified recombinant NS5B was incubated with the activated recombinant Akt-1 protein or BSA in the kinase buffer containing γ-32P ATP or excess cold ATP. A signal appeared only in reaction containing NS5B-2b but disappeared after thrombin digestion, suggesting the signal might come form nonspecific phosphorylation on protein kinase A site which is at upstream of NS5B. Unequal amount of NS5B-1b and NS5B-2b might be another reason for this false-positive result. To eliminate this concern, we used the M2 resin to capture the recombinant NS5B proteins and quantitated the captured NS5B proteins by western blot before thrombin digestion. Equal mount of NS5B-1b and NS5B-2b protein, with or without thrombin digestion, were applied for Akt kinase assay. By using the immuno-complex Akt kinase assay, specific phosphorylation signal appeared in reaction containing NS5B-1b but not NS5B-2b. The result clearly demonstrated that NS5B could be phosphorylated by Akt, indicating that HCV NS5B is a novel substrate of Akt. The result may have implication in pathogenesis of HCV replication and novel treatment development for HCV infection.

Abstract in Chinses………………………………………………1

Abstract in English………………………………………………2-3

I.Introduction …………………………………………………4-7

II.Materials and Methods ………………………………………8-15

(1) E. coli expression system………………………………9

(2) Baculovirus expression system…………………………9-12

(3) ÄKTAprimeTM system ………………………………………12-14

(4) Immuno-precipitation ……………………………………14-15

(5) Akt kinase assay …………………………………………15

III.Results…………………………………………………………16-19

(1) E. coli expression system………………………………16-17

(2) Baculovirus expression system…………………………17-18

(3) Akt kinase assay …………………………………………18-19

IV.Discussion………………………………………………………20-23

V.Figures……………………………………………………………24-40

VI.References………………………………………………………41-42

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