端粒酶基因的腺病毒包裝條件優化及其活性功能驗證

賀小英 荊乾鴿 姜欣穎 袁婷 吳憲 佟瑞瑩 王晶妍 馬利兵

摘要：【目的】構建攜帶人類端粒酶催化亞基基因（hTERT）的腺病毒載體并優化其包裝條件，為進一步探究端粒酶與細胞重編程調控間的作用機理提供參考依據。【方法】利用腺病毒載體pAdEasy-1構建攜帶hTERT基因的重組腺病毒載體，分別采用脂質體法和電轉法及不同濃度胎牛血清在HKE293A細胞中進行包裝，篩選出最佳病毒包裝條件，收集重組腺病毒rAd-hTERT并體外感染小鼠胎兒成纖維細胞，驗證重組腺病毒rAd-hTERT的活性功能。【結果】以腺病毒載體pAdEasy-1為骨架質粒，成功構建獲得攜帶hTERT基因的重組腺病毒載體（pAd-hTERT），其最佳病毒包裝條件是采用脂質體法結合15%胎牛血清，此條件下收集獲得的病毒滴度最高（1×1011 IFU/mL）。以攜帶hTERT基因的腺病毒rAd-hTERT感染小鼠胎兒成纖維細胞72 h后，可擴增出片段大小約3450 bp的目的基因條帶，即hTERT基因在小鼠胎兒成纖維細胞中成功表達。【結論】采用脂質體法結合15%胎牛血清包裝獲得的攜帶hTERT基因重組腺病毒具有較高病毒滴度，感染小鼠胎兒成纖維細胞能成功表達hTERT基因，進一步證實端粒酶具有蛋白功能的生物學活性。

關鍵詞： 端粒酶;腺病毒;細胞重編程;載體構建;病毒包裝;功能驗證

中圖分類號： S814.8? ? ? ? ? ? ? ? ? ? ? 文獻標志碼： A 文章編號：2095-1191（2019）06-1356-06

Abstract：【Objective】The aim of the study was to construct an adenoviral vector containing the telomerase catalytic subunit（hTERT） gene， optimize its packaging conditions and lay a foundation for further study of the interaction mechanism between telomerase and cell reprogramming. 【Method】The adenoviral vector containing the hTERT gene was constructed by using the AdEasy-1 system of recombinant adenovirus， and then it was packaged in HKE293A cells by liposome， electroporation and different serum concentrations of fetal bovine. The optimal packaging condition was selected. Recombinant adenovirus rAd-hTERT was collected and infected mouse fetal fibroblasts in vitro， and activity function of recombinant adenovirus rAd-hTERT was verified. 【Result】The recombinant adenoviral vector（pAd-hTERT） carrying hTERT gene was successfully constructed with recombinant adenovirus rAd-hTERT as skeleton plasmid. The optimal packaging condition was liposome method combining with 15% serum of fetal bovine. The titer of virus obtained under such condition was the highest（1×1011 IFU/mL）. After 72 h of adenovirus（rAd-hTERT） carrying hTERT gene infected mouse fetal fibroblasts， target gene bands of 3450 bp was amplified， which meaned that hTERT gene was successfully expressed in mouse fetal fibroblasts. 【Conclusion】The recombinant adenovirus carrying hTERT gene has been successfully prepared by liposome method combining with 15% serum of fetal bovine. It has high virus titer. Infecting mouse fetal fibroblasts can successfully express hTERT gene， which further verifies that telomerase has biological activity of functional protein.

Key words： telomerase; adenovirus; cell reprogramming; vector construction; virus packaging; functional verification

收稿日期：2019-04-04

基金項目：國家自然科學基金項目（31560331，31660340）;內蒙古自然科學基金項目[2017MS（LH）0213，2017MS0337]

作者簡介：賀小英（1978-），博士，副教授，主要從事動物細胞重編程及轉基因技術研究工作，E-mail：hxy1124@163.com

0 引言

【研究意義】端粒酶（Telomerase）是一種特殊的逆轉錄酶，能通過延長端粒進而參與細胞周期調控，而端粒酶基因是研究細胞衰老、癌癥致病機理及制備永生化細胞系的關鍵基因（Le?o et al.，2018;Spiegl-Kreinecker et al.，2018）。……