摘要
目的 构建并鉴定携带大鼠线粒体融合蛋白（rMfn2）基因的重组腺病毒AdS-mCMV-增强绿色荧光蛋白（EGFP）-CMV-rMfn2表达载体.方法 设计含有Not Ⅰ和HindⅢ酶切位点的引物,应用聚合酶链反应（PCR）法扩增目的基因rMfn2,将扩增产物连接到穿梭质粒上,构建重组穿梭质粒pDC316-mCMV-EGFP-CMV-rMfn2.测序鉴定重组穿梭质粒后,与腺病毒骨架质粒pAd5在JM109感受态细胞中同源重组,获得重组腺病毒AD-rMfn2,而后感染HEK293细胞进行包装、扩增、纯化,并测定病毒滴度;逆转录-聚合酶链反应（RT-PCR）及Western blot检测目的基因rMfn2在HEK293细胞中的转录及表达.结果 腺病毒穿梭质粒经PCR、双酶切及测序鉴定证明构建正确,目的基因rMfn2正确克隆至腺病毒骨架质粒中;在HEK293细胞中包装、扩增获得了高滴度的重组腺病毒AD-rMfn2,其滴度为4×1013 TU/L;AD-rMfn2感染HEK293细胞48 h后,可检测到目的基因rMfn2的转录和蛋白表达.结论 成功构建重组腺病毒AD-rMfn2.
Objective To construct and identify the recombinant adenovirus carrying rat mitochondria fusion （rMfn2） gene,providing a foundation for further research.Methods With a pair of primers containing Not Ⅰ and Hind Ⅲ restriction sites,the polymerase chain reaction （PCR）-amplified products of rMfn2 were subcloned into shuttle plasmid to obtain pDC316-mCMV-enhanced green fluorescent protein （EGFP）-CMV-rMfn2.The constructed recombinant plasmid was identified by sequencing and used for homologous recombination with adlenovirus backbone plasmid pAd5 in competent cells JM109.HEK293 cells were transfected withthe acquired recombinant adenovirus AD-rMfn2 for packaging,propagation and purification,and the virus titer was determined.Teverse transcriptase （RT）-PCR and Western blotting were used to detect the transcription and expression of rMfn2 respectively.Results PCR,restriction analysis and sequencing validated that adenovirus shuttle plasmid pDC316-mCMV-EGFP-CMV-rMfn2 was constructed correctly.rMfn2 was cloned into adenovirus backbone plasmid pAd5 correctly.After packaging and propagation in HEK293 cells,recombinant adenovirus with a titer of 4 × 1013 TU/L was obtained.The transcription and expression of rMfn2 gene were proved in HEK293 cells after infection with the recombinant adenovirus in 48 h.Conclusion Recombinant adenovirus vector containing rMfn2 has been constructed successfully,which established a basis for further research.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第2期295-297,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81160037）
海南省引进集成应用项目（YJJC20120006）
海南省自然科学基金资助项目（812202）
关键词
线粒体融合蛋白基因
重组腺病毒
Mitochondria fusion protein gene
Recombinant adenovirus
