人FRNK重组腺病毒的构建

目的:应用AdEasy复制缺陷型腺病毒载体系统构建人FRNK基因重组腺病毒,并在HEK293细胞中扩增制备重组病毒。方法:把人FRNK基因克隆入腺病毒穿梭载体pAdTrack-CMV中,构建腺病毒质粒pAdTrack-CMV-hFRNK,经PmeⅠ内切酶酶切成线性化后,采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中,通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到HEK293细胞进行包装,获得人FRNK重组腺病毒pAdhFRNK。荧光显微镜下观察绿色荧光蛋白的表达。结果:经酶切和绿色荧光蛋白表达证明了hFRNK基因的重组腺病毒载体pAdhFRNK构建成功,并制备出高滴度的重组病毒。结论:成功构建携带人FRNK基因的重组腺病毒pAdhFRNK,为研究FRNK的基因治疗奠定了基础。

Construction of Human FRNK Recombinant Adenovirus Vector

Objective: To construct an hFRNK adenovirus vector,pAdhFRNK.Methods: Human FRNK was cloned into shuttle vector pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-hFRNK,and the resultant plasmid was linearized by Pme Ⅰ enzymatic digestion and was subsequently electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.Recombinants were screened by Kanamycin and were identified by observation of green fluorescent protein(GFP) expression and PCR.The Pac Ⅰ-linearized recombinant plasmid was transfected into HEK293 cells to package the adenovirus,and the hFRNK recombinant adenovirus was observed by fluorescent microscope.Results: The hFRNK recombinant adenoviral vector was constructed successfully,which was confirmed by restriction enzyme and GFP expression.Conclusion: The human FRNK recombinant adenovirus was constructed successfully,and provided a sound base for gene therapy.