一种改进的重组腺病毒载体的包装和克隆纯化方法

目的:重组腺病毒载体是目前使用最多的病毒载体之一。常用的AdEasyTM包装系统在制备条件复制型腺病毒时易混有野生型腺病毒或可复制型腺病毒(RCA),在HEK293细胞中包装出的重组腺病毒必须经过2～3轮噬斑的筛选,费时费力。本实验拟对常规包装方法进行改进。方法:将腺病毒载体质粒转染至HEK293细胞过程中的液体培养基更换为琼脂糖凝胶的混合培养液,因为凝胶形成的阻碍,包装出的病毒不能通过细胞-培养液-细胞的方式进行扩散,只能依靠细胞-细胞的方式来扩展而形成病灶(噬斑);然后随机挑选单个噬斑进行PCR鉴定,筛选出特异的目标病毒。结果:采用常规方法在HEK293细胞中初包装出的腺病毒,同源重组产生的RCA已被检测到。采用改进方法制备的重组腺病毒,能排除背景病毒的干扰,使重组腺病毒载体的包装和克隆纯化一步完成;利用此病毒初步在体外实现了对人肝癌细胞的特异性杀伤。结论:改进的方法是一种高效、简便、快捷地包装并纯化重组溶瘤腺病毒的方法,采用该方法包装的重组溶瘤腺病毒能满足实验的需要。

An Optimized Method for Packaging and Cloning Purification of Recombination Adenovirus

Objective: Recombination adenoviral vector is one of the most common tools in gene therapy.AdEasyTM adenoviral vector system,which is often used to produce the conditional replication-competent adenovirus,is always mixed with native adenovirus or replication-competent adenovirus(RCA),and it needs twice or thrice plaque screening procedure to purify the recombination adenovirus in HEK293 cells.That might be time-consuming and work labored.This study is to optimize the conventional method of recombination adenovirus packaging.Methods: The liquid growth medium used in transfection adenovirus vector plasmid into competent cell HEK293 cells was substituted by agarose mixture growth medium.Since agarose-gel inhibited the spread of packaged adenovirus in cell-growth medium-cell pattern,the virus expansion was in cell-cell pattern to from plaque.The isolated plaques were cored-out randomly followed by PCR identification in order to screen the specific desired adenovirus.Results: There was a detectable homologous recombination between the E1-deleted vector and the host DNA resulting in the production of some RCA in primary virus stock by conventional packaging method.Whereas the optimized method avoided the interference of RCA and the recombinant adenovirus packaging and cloning purification proceeded in one-step.Conclusion: It is a simple,rapid and high efficient method for the packaging and cloning purification of recombination adenovirus,which meet the demands of subsequent experiments.