本文選題：重組腺病毒 + 嵌合　； 參考：《吉林大學》2012年碩士論文

【摘要】：重組5型腺病毒載體已被廣泛應用于人類免疫缺陷病毒（HIV）、乙肝病毒、結(jié)核桿菌等疫苗和腫瘤基因治療的研究。然而，人體內(nèi)普遍存在的針對載體本身的預存免疫會顯著地降低病毒載體的轉(zhuǎn)導效率和轉(zhuǎn)基因表達水平。本論文首先根據(jù)目前對57種亞型的人腺病毒的血清陽性率調(diào)查結(jié)果，然后通過分析所有稀有型腺病毒六鄰體超變區(qū)（HVR）的基因和氨基酸序列，選擇出理想的替換血清型Ad26，Ad37和Ad43。再次，結(jié)合相關(guān)文獻報道的將稀有亞型人腺病毒六鄰體與Ad5進行替換的研究結(jié)果，，設(shè)計以Ad26，Ad37和Ad43的全長HVR5和HVR7基因以及Loop4基因替換rAd5相應區(qū)域來構(gòu)建六鄰體嵌合型rAd5載體。 我們最終成功構(gòu)建了6種六鄰體嵌合型rAd5載體rAd5HVR26(5+7)、rAd5HVR26(5+7)Loop4、 rAd5HVR37(5+7)、 rAd5HVR(5+7)37Loop4、rAd5HVR43(5+7)和rAd5HVR43(5+7)Loop4。為方便對比評價嵌合載體的包裝、復制能力以及轉(zhuǎn)基因傳代穩(wěn)定性和表達水平，我們將GFP和Luciferase報告基因分別克隆到穿梭載體pDC316上。與改造的嵌合腺病毒載體共轉(zhuǎn)293cell和Hela細胞，通過熒光顯微鏡下綠色熒光的觀察或Luciferase活性檢測來確認嵌合載體的相關(guān)性質(zhì)。 本論文最終得到了一種有利于包裝，傳代性質(zhì)穩(wěn)定，并且復制能力和轉(zhuǎn)基因表達水平都顯著提高的嵌合載體Ad5HVR43(5+7)，為后續(xù)在體內(nèi)實驗中評價其免疫水平，探索腺病毒免疫機制打下基礎(chǔ)，期望得到免疫水平較高且能很好的避免預存免疫的重組腺病毒載體，以應用于腫瘤基因治療和人類免疫缺陷病毒、結(jié)核等疫苗的研究。本論文的研究對于腺病毒載體修飾后的包裝有一定意義，同時也為腺病毒載體的進一步修飾改造提供了重要依據(jù)。
[Abstract]:Recombinant adenovirus vector 5 has been widely used in the study of human immunodeficiency virus (HIV), hepatitis B virus (HBV), tuberculosis vaccine and tumor gene therapy. However, the preexisting immunization against the vector itself can significantly reduce the transduction efficiency and transgenic expression level of the virus vector. In this paper, the seropositive rate of 57 subtypes of human adenovirus was investigated. Then, by analyzing the gene and amino acid sequence of all rare adenovirus, the ideal replacement serotypes Ad26AAd37 and Ad43were selected. Thirdly, based on the reported results of replacing the rare subtype of human adenovirus with Ad5, we designed to replace the rAd5 region with the full-length HVR5 and HVR7 genes of Ad26Ad37 and Ad43 and Loop4 gene to construct the hexagonal chimeric rAd5 vector. We successfully constructed six kinds of hexagonal chimeric rAd5 vectors, rAd5HVR26(5 7rAd5HVR2657 Loop4, rAd5HVR37(5 7, rAd5HVR(5 7 7Loop4, rAd37Loop4 rAd5HVR4357) and rAd5HVR43(5 7 Loop4. In order to compare and evaluate the packaging, replication ability, stability and expression level of chimeric vectors, GFP and Luciferase reporter genes were cloned into shuttle vector pDC316 respectively. The chimeric vector was co-transfected with the modified chimeric adenovirus vector to 293cell and Hela cells. The properties of the chimeric vector were confirmed by the observation of green fluorescence under fluorescence microscope or the detection of Luciferase activity. In this paper, a chimeric vector, Ad5HVR43(5 7, which is favorable to packaging, stable in passage, and significantly improved in replication and transgenic expression, was obtained to evaluate its immune level in vivo. To explore the immune mechanism of adenovirus, we hope to obtain recombinant adenovirus vector with high immune level and avoid preexisting immunity, which can be used in tumor gene therapy, human immunodeficiency virus, tuberculosis and other vaccines. The research in this paper has some significance for the packaging of adenovirus vector, and also provides an important basis for the further modification of adenovirus vector.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.11

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本文編號：1929722