透過Qβ-VLPmRNA表達功能性蛋白之探討

Abstract

在迅速發展的核酸治療方法中，相較於去氧核醣核酸（DNA）療法，以訊息核糖核酸（mRNA）攜帶基因訊息並表達特定蛋白質的方法提供了一個更具優勢的研究選擇。透過mRNA進行的療法能夠提高標的基因的轉譯效率，並且降低插入式誘導突變所帶來的潛在風險。然而，訊息核糖核酸的應用仍受限於其不穩定性以及製作訊息核糖核酸所需的生產成本。因此，為了實踐核糖核酸的應用，建立一個穩健的核糖核酸運輸系統是必要的。一個理想的核糖核酸運送系統須符合幾個條件：(1)能夠保護核糖核酸不受到普遍存在的核酸內切酶所降解、(2)防止核糖核酸與非特定之生物分子進行交互作用、(3)擁有針對特定細胞或組織運輸核糖核酸之能力以及(4)促進核糖核酸進入細胞之效率。 類病毒蛋白顆粒（VLPs）是由非感染性之病毒外殼蛋白以高度重複性結構構成的大分子。由病毒衍生而來的類病毒蛋白顆粒能夠在生物體被表達，並且能夠藉由該表達系統，將病毒殼蛋白(coat protein, CP)自動組裝成完整的類病毒蛋白顆粒。在過去的研究中，已有科學家們利用類病毒蛋白顆粒的自動組裝性質包裝功能性生物大分子，例如蛋白質與核酸。在此，我們選擇以尿酸氧化酶（UOX）作為研究主題。尿酸氧化酶是一種催化尿酸氧化反應的酵素，在大多數哺乳動物體內負責調節尿酸的恆定。在此研究中，我們希望藉由類病毒蛋白顆粒的自組裝性質以包裝尿酸氧化酶的訊息核糖核酸，並利用類病毒蛋白顆粒探討功能性蛋白於真核系統中表達的可行性。 目前，我們已經成功地將尿酸氧化酶以及Qβ病毒外殼蛋白（Qβ-CP）的基因構建到質體pT7CFE1-NHA-UOX和pCDF-1b-CP中，並將這兩種質體共同轉形至大腸桿菌品系BL21以表達包裝尿酸氧化酶之訊息核糖核酸的類病毒蛋白顆粒Qβ-VLPmUOX。我們首先透過穿透式電子顯微鏡觀察Qβ-VLPmUOX之組裝情形，並利用變性凝膠電泳確認包裝在Qβ-VLPmUOX中的訊息核糖核酸(cargo transcripts)。我們也利用無細胞轉譯(cell-free translation)進行尿酸氧化酶的蛋白表達，並驗證我們設計的質體能夠透過轉錄以及轉譯得到具有功能的尿酸氧化酶。在透過AmplexRed試劑對無細胞轉譯之產物探討酵素活性的結果中顯示:利用質體作為起始物所產出的尿酸氧化酶具有酵素活性；當使用包裝在Qβ-VLPmUOX中的尿酸氧化酶的訊息核糖核酸作為起始物時的無細胞轉譯產物則偵測不到其酵素活性。此外，針對Qβ-VLPmUOX在真核細胞中表達尿酸氧化酶的初步實驗也未偵測到尿酸氧化酶產出的存在。因此，目標利用Qβ-VLPmRNA在生物體中表達功能性蛋白直至目前仍具挑戰，於本論文中所討論已知的實驗困難則需進一步的研究與克服。

Among rapidly developed nucleic acid therapeutics, mRNA-mediated approach offers an attractive alternative to DNA-mediated gene therapy. The mRNA-based approaches have potential to increase translation efficiency and to lower insertional mutagenesis compared that to DNA-based therapies. However, the applications of mRNA are limited to its instability and the cost to produce. Therefore, a robust RNA delivery system is required for achieving RNA applications. An ideal RNA delivery system is expected to (i) protect cargo against ubiquitous nucleases, (ii) prevent un-wanted interaction with non-specific biomolecules, (iii) deliver cargo to tissues of interest, and (iv) promote cell entry efficiency. Virus-like particles (VLPs) are macromolecular assemblies of non-infectious viral coat proteins (CP) in a highly ordered repetitive structural configuration. The VLPs derived from virus counterparts can be expressed in vivo and CPs are able to assemble into a complete particle spontaneously. Prior art VLPs are found being capable of packaging biological macromolecules such as proteins and nucleic acids. Here we use uricase (UOX) as a study case in this research. The UOX is an enzyme that catalyzes the oxidation reaction of uric acid and also regulates uric acid homeostasis in most mammalians. Here we propose to use VLPs to encapsulate the UOX messenger RNA (mRNA), deliver it to host cells via VLPs, and explore the feasibility of functional protein expression in eukaryotic systems. In this study, the UOX and Qβ-CP genes are successfully designed and cloned into two plasmids (pT7CFE1-NHA-UOX and pCDF-1b-CP). These two constructs were co-transformed into E. coli strain BL21 to produce Qβ-VLPmUOX. We validated the assembly of the Qβ-VLPmUOX by using TEM technique. The cargo transcripts (mUOX_P and mUOX_SP) packed inside Qβ-VLPmUOX is verified by the denaturing PAGE. In addition, we showed that the cargo plasmid is capable of being transcribed and then translated into a functional UOX by utilizing the cell-free translation. The function of the in vitro expressed UOX is assayed positively via the AmplexRed uricase assay. Meanwhile, the Qβ-VLP-encapsulated mUOX shows no translational capability neither in cell-free translation system nor in cell culture despites the input mass of the cargo transcripts varies up to 2000 g. The goal of using Qβ-VLPmRNA for functional protein production in vivo remains a challenge and yet some obstacles discussed in the contents require to be further investigated and overcome in future work.