一种简易、廉价、高效构建重组腺病毒载体的方法

Construction of recombinant adenoviral vector by a simple, cheap,and high performance method

目的:建立构建重组腺病毒载体的简易、廉价、高效方法,为进一步应用含自杀基因的重组腺病毒治疗恶性肿瘤奠定基础。方法:PmeⅠ线性化腺病毒穿梭质粒pAdTrack-CMV、切胶纯化,与腺病毒骨架质粒pAdEasy-1按一定比例混合后,以化学转化法导入大肠杆菌BJ5183,在菌内进行同源重组,构建重组腺病毒质粒rpAdEasyGFP。以脂质体法将重组质粒转染293细胞包装重组腺病毒rAdGFP。结果:细菌内同源重组率高达80%;在293细胞中成功产生重组腺病毒。结论:对目前应用广泛的AdEasy系统进行了有效改进,在获得较高阳性重组率的同时,降低了对实验室设备的要求。

Objective To set up a basis for treatment of malignant tumor with recombinant adenovirus carrying suicide genes establish a simple cheap and high efficient method of construction of recombinant adenoviral vector. Methods Adenoviral shuttle plasmid pAdTrack-CMV was linearized by PmeⅠand then purified by gel. Mixed the purified digest and adenoviral backbone plasmid pAdEasy-1 with proper proportion and transformed them into E.coli BJ 5 183 with chemical transformation method. In BJ 5 183 homologous recombination was occurred and recombinant adenoviral plasmid rpAdEasyGFP was generated. Recombinant plasmid was then transfected into 293 cells by liposome method to package recombinant adenovirus rAdGFP. Results The rate of homologous recombination was up to 80%. Recombinant adenovirus was produced in 293 cells successfully. Conclusion AdEasy system a broad applied method presently was improved. The new method can obtain higher positive recombinant rate as well as reduce the requirement for experimental equipments.