本文關鍵詞：FoxM1相關重組腺病毒載體小試生產及初步質量控制研究　出處：《湖南大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 基因治療 重組腺病毒 FoxM1 超濾 離子交換 凝膠過濾

【摘要】：基因治療技術最初作為遺傳病的一種治療手段開始被人們熟知。二十多年來,隨著基因治療技術的發(fā)展進步,基因治療已經被應用于多種疾病的治療,尤其是腫瘤的基因治療。據(jù)全球基因治療臨床數(shù)據(jù)庫的數(shù)據(jù)顯示,目前全球腫瘤基因治療應用范例中使用最多的載體是腺病毒載體。隨著基因治療向臨床轉化的深入,對基于腺病毒載體的藥物需求日益增大,質量要求也增高,因此大規(guī)模臨床級別腺病毒載體藥物的生產成為一個難題。傳統(tǒng)純化腺病毒的氯化銫密度梯度離心的方法已經不能滿足需求,目前層析方法純化腺病毒已成為規(guī)模生產的首選方法。重組腺病毒AdFoxM1shRNA是在FoxM1可作為抑制腫瘤生長的靶基因的基礎上研發(fā)的新型重組腺病毒藥物載體。本研究中我們即利用了AdFoxM1shRNA進行了腺病毒的小試生產和初步質量控制研究。通過引入生產時需構建細胞庫的概念,建立了293A細胞的細胞庫,確保了后續(xù)產品來源的穩(wěn)定性和同一性。并運用轉瓶培養(yǎng)體系擴增293A細胞,進行病毒感染,收集病毒擴增時的上清液。在下游純化工藝研究中通過摸索超濾濃縮、陰離子交換層析和凝膠過濾層析等工藝獲得了大量的AdFoxM1shRNA重組腺病毒,最后通過純度、病毒滴度、復制型病毒檢測三個核心指標對獲得的重組腺病毒進行了初步質量控制研究,并與傳統(tǒng)方法獲得的重組腺病毒載體進行對比,確定層析方法獲得的AdFoxM1shRNA重組腺病毒質量的優(yōu)越性。
[Abstract]:Gene therapy has been widely known since 20 years. With the development of gene therapy, gene therapy has been used in many diseases. In particular, gene therapy for cancer. According to the global database of gene therapy clinical data shows. At present, adenovirus vector is the most widely used vector in the application of gene therapy in the world. With the further transformation of gene therapy to clinic, the demand for the drug based on adenovirus vector is increasing day by day, and the quality requirement is also increasing. Therefore, the production of large clinical grade adenovirus vector drugs has become a difficult problem. The traditional method of cesium chloride density gradient centrifugation for the purification of adenovirus can not meet the demand. At present, purification of adenovirus by chromatography has become the preferred method for large-scale production. Recombinant adenovirus AdFoxM1shRNA is a new type of adenovirus based on FoxM1 as a target gene to inhibit tumor growth. Recombinant adenovirus drug vector. In this study, we used AdFoxM1shRNA to carry out the pilot production of adenovirus and preliminary quality control study. By introducing the concept of construction of cell bank during production. The cell bank of 293A cells was established to ensure the stability and identity of the subsequent product source. 293A cells were amplified by flask culture system for virus infection. The supernatant of virus amplification was collected and concentrated by ultrafiltration in the downstream purification process. A large number of AdFoxM1shRNA recombinant adenovirus were obtained by anion exchange chromatography and gel filtration chromatography. Finally, the purity and titer of the recombinant adenovirus were obtained. The quality control of the recombinant adenovirus was studied by three core indicators of replicative virus detection and compared with the recombinant adenovirus vector obtained by the traditional method. To determine the superiority of AdFoxM1shRNA recombinant adenovirus quality obtained by chromatography.
【學位授予單位】：湖南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

【參考文獻】

相關期刊論文 前4條

1 王振發(fā);王烈;衛(wèi)立辛;;基因治療病毒載體的研究進展[J];福州總醫(yī)院學報;2009年04期

2 祁麗,顧銘,叢威;重組腺病毒生產技術研究進展[J];過程工程學報;2004年05期

3 田博;吳彬;張群偉;畢建進;王瀾;朱寶珍;耿越;吳祖澤;;懸浮培養(yǎng)HEK-293 N3S細胞生產重組腺病毒Ad-GFP的實驗研究[J];生物工程學報;2007年05期

4 王曉娜;肖鳳君;吳彬;吳祖澤;畢建進;張以芳;;重組腺病毒Ad-GFP的純化[J];生物技術通訊;2009年06期

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本文編號：1365259