大鼠重组腺病毒载体RP105的构建和鉴定

Construction and identification of rat recombinant adenovirus vector RP105

目的构建和鉴定大鼠RP105重组腺病毒载体,并获得具有一定滴度的重组腺病毒表达载体Ad-RP105-EGFP。方法 PCR钓取目的基因RP105,连接入穿梭载体GV135(CMV-MCS-EGFP),形成连接产物。连接好的重组腺病毒载体CMV-RP105-MCS-EGFP转化大肠埃希菌DH5a感受态细胞获得大量阳性克隆,对阳性克隆进行酶切鉴定及测序鉴定。采用AdMax腺病毒包装系统,将成功构建的RP105重组腺病毒穿梭质粒与辅助包装质粒共转染HEK293细胞,通过Cre/loxP重组酶系统的作用实现重组,得到重组腺病毒,并进行重组腺病毒扩增、纯化及滴度测定。结果重组腺病毒穿梭质粒酶切鉴定与测序证明本实验成功构建了大鼠RP105重组腺病毒载体。RP105穿梭质粒和腺病毒包装质粒共转染HEK293细胞24h后可见大量绿色荧光蛋白表达和聚集,10d后出现典型的细胞病变,说明腺病毒包装成功。再经重组腺病毒扩增、纯化及滴度测定,重组腺病毒滴度为1×109 PFU/ml。结论本实验成功构建了携带RP105基因的重组腺病毒表达载体Ad-RP105-EGFP。这为下一步研究其对心肌缺血再灌注损伤的保护作用及分子机制奠定了实验基础。

Objective To establish the recombinant adenovirus expression vector Ad-RP105-EGFP by constructing and identifying the rat recombinant adenovirus vector RPI05. Methods The targeting gene RP105 was fished by PCR and linked to the shuttle vector GV135 （CMV-MCS- EGFP） to form a linker product. A large number of positive clones were produced by transforming the recombinant vector CMV-RP105-MCS-EGFP to E. coli DHSa cells,and confirmed by enzyme digestion and DNA sequencing. HEK293 cells were cotransfected to the recombinant shuttle vec- tor RP105 and auxiliary packaging plasmids using the AdMax virus packaging system. The recom- binant adenovirus vector was established using the Cre/loxP recombinant enzyme system, ampli- fied and purified with its titer measured. Results Enzyme digestion and DNA sequencing of the recombinant shuttle vector showed that the rat recombinant adenovirus vector RP105 was suc- cessfully constructed with a titer of 1 ×10^9 PFU/ml after packaging, amplification and purifica- tion. Conclusion The recombinant adenovirus vector Ad-RP105-EGFP we constructed in this study lays a foundation for further studying its role and molecular mechanism in protecting myo- cardium against ischemic reperfusion injury.