仅有部分慢病毒载体在宿主细胞内发生了功能性整合

来源 :第十四次全国医学遗传学学术会议 | 被引量 : 0次 | 上传用户:fuzhi2009
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  目的:慢病毒载体是以慢病毒为基本骨架,经功能性删减和拆分制备而成只具备一次性感染和整合能力的病毒载体.因此,理论上慢病毒载体的基本生理特性应遗传了其来源病毒的特征.一般认为,慢病毒在感染细胞后,仅有部分骨架是以整合入染色体的形式存在的.然而,慢病毒载体是否遗传了这一特点未见文献报导.本文旨在研究慢病毒载体在感染细胞后,是否全部整合入染色体.方法:用慢病毒载体的假病毒感染293T细胞后,通过细胞传代来稀释未整合的慢病毒载体原病毒.在此基础上,分别抽提培养2天和10天(分裂了103次)的细胞基因组DNA.通过定量PCR检测细胞基因组中的LTR拷贝数,计算出整合率.分别取病毒感染3天和11天的细胞,离心重悬后,取部分细胞放在荧光显微镜下拍照,在蛋白水平上验证此结论.结果:慢病毒载体在宿主细胞基因组内的平均整合率为47±16%(n=10).细胞感染慢病毒载体假病毒3天和11天以后的GFP照片也证实了慢病毒载体在293T宿主细胞基因组内部分整合.结论:慢病毒载体的假病毒感染293T细胞后,仅有部分的载体骨架是整合入基因组中的,整合入293T细胞基因组中的外源基因能够较长时间地稳定表达.
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