CAJ | 학술논문

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.
목적 구건병감정휴대대서선립체융합단백(rMfn2)기인적중조선병독AdS-mCMV-증강록색형광단백(EGFP)-CMV-rMfn2표체재체.방법 설계함유Not Ⅰ화HindⅢ매절위점적인물,응용취합매련반응(PCR)법확증목적기인rMfn2,장확증산물련접도천사질립상,구건중조천사질립pDC316-mCMV-EGFP-CMV-rMfn2.측서감정중조천사질립후,여선병독골가질립pAd5재JM109감수태세포중동원중조,획득중조선병독AD-rMfn2,이후감염HEK293세포진행포장、확증、순화,병측정병독적도;역전록-취합매련반응(RT-PCR)급Western blot검측목적기인rMfn2재HEK293세포중적전록급표체.결과 선병독천사질립경PCR、쌍매절급측서감정증명구건정학,목적기인rMfn2정학극륭지선병독골가질립중;재HEK293세포중포장、확증획득료고적도적중조선병독AD-rMfn2,기적도위4×1013 TU/L;AD-rMfn2감염HEK293세포48 h후,가검측도목적기인rMfn2적전록화단백표체.결론 성공구건중조선병독AD-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.