人IL-24基因重组腺病毒的构建及其在A549细胞中的表达  

Construction of recombinant adenovirus containing human IL-24 gene and expression in A549 cells

作　　者：田文标[1] 陈丽芬[1] 邹全明[1] 

机构地区：[1]第三军医大学医学检验系临床微生物与免疫学教研室,重庆400038

出　　处：《第三军医大学学报》2005年第21期2115-2117,共3页Journal of Third Military Medical University

摘　　要：目的构建含有人IL-24基因的重组腺病毒载体,并转染肺腺癌细胞A549细胞观察其感染能力,为进一步的基因治疗奠定实验基础。方法采用基因工程技术将人IL-24基因cDNA亚克隆至穿梭质粒pAdTrack-CMV上,采用PAdEasy系统进行细菌内同源重组,后通过脂质体将正确重组体包裹并转染293T细胞以包装并扩增病毒。采用PCR方法对重组腺病毒进行鉴定,利用穿梭质粒中所带绿色荧光蛋白GFP报告基因,对病毒滴度和感染效率进行检定;最后采用免疫组化法对IL-24在A549中的表达进行检测。结果酶切和PCR结果证实IL-24基因重组腺病毒载体构建成功,病毒滴度达1.2×1010pfu/ml,能够成功转染A549细胞并在其中进行表达。结论成功构建了有较强感染能力的含人IL-24基因的重组腺病毒载体。Objective To construct the recombinant adenovirus encoding human IL-24 gene for future gene therapy. Methods The human IL-24 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction （PCR）. The titer and its infection rate were determined using the green fluorescent protein （GFP） expression in the shuttle plasmid. The expression of target protein was measured by the method of immunohistochemistry. Results Restriction endonuclease and PCR analysis confirmed that the human IL-24 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.2×10^10pfu/ml. The adenovirus has a strong effect on A549 cells and human IL-24 can express in it. Conclusion The recombinant adenovirus containing human IL-24 gene was successfully constructed by the method of homogenous recombination in bacteria.

关 键 词：腺病毒 L-24基因 源重组 

分 类 号：R373.1[医药卫生—病原生物学] R392.2[医药卫生—基础医学]