抑瘤细胞运动和侵袭的鼠FRNK重组腺病毒的构建

Construction of inhibiting tumor cell motility and invasion murine FRNK recombinant adenovirus vector

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 a FRNK adenovirus vector, pAdFRNK . Methods Murine FRNK was cloned into shuttle vector pAdTrack-CMV to generate a recombinant plasmid pAdTrackCMV-FRNK,and the resultant plasmid was linearizcd by Pme I enzymatic digestion and subsequently electroporated into E. coli BJ5183 cells containing adenovirus backbone plasmid pAdEasy-1. Recomhinants were screened by Kanamycin and the recombinant adenovirus identified by observation of green fluorescent protein expression and PCR. The Pac I-linearized identified recombinant plasmid was transfected into 293HEK cells to package the adenovirus. At last FRNK recombinant adenovirus production was observed by fluorescent microscopy. Results A FRNK recombinant adenoviral vector was constructed successfully, which was confirmed by restriction enzyme and GFP expression. Conclusion The murine FRNK re- combinant adenovirus were constructed successfully,which provided a sound base for gene therapy.