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目的应用AdEasy复制缺陷型腺病毒载体系统构建小鼠FRNK基因重组腺病毒,并在293HEK细胞中扩增制备重组病毒。方法把小鼠FRNK基因克隆入腺病毒穿梭载体pad- Track-CMV中,构建腺病毒穿梭质粒pAdTrack-CMV-FRNK,经Pme I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293HEK细胞进行包装,获得FRNK重组腺病毒pAdFRNK。荧光显微镜下观察绿荧光的表达。结果经酶切和绿荧光表达证明了小鼠FRNK基因的重组腺病毒载体pAdFRNK构建成功,并制备出高滴度的重组病毒。结论成功构建携带小鼠FRNK基因的重组腺病毒pAdFRNK,为研究FRNK的基因治疗奠定了基础。
Objective To construct the recombinant adenovirus of mouse FRNK gene by AdEasy replication-deficient adenovirus vector system and amplify the recombinant adenovirus in 293HEK cells. Methods Mouse FRNK gene was cloned into the adenovirus shuttle vector pad-Track-CMV to construct adenovirus shuttle plasmid pAdTrack-CMV-FRNK. After digested with Pme I endonuclease, the recombinant plasmid was transformed into To competent E. coli BJ5183 containing adenovirus backbone vector pAdEasy-1, positive recombinant adenovirus was obtained by kanamycin screening. The recombinant adenovirus plasmid was transfected into 293HEK cells for packaging, and the FRNK recombinant adenovirus pAdFRNK was obtained. Fluorescence microscopy was used to observe the expression of green fluorescence. Results The recombinant adenoviral vector pAdFRNK of FRNK gene was successfully constructed and the recombinant virus with high titer was successfully constructed. Conclusion The recombinant adenovirus pAdFRNK carrying mouse FRNK gene was successfully constructed and laid the foundation for the study of gene therapy of FRNK.