CAJ | 학술논문

目的构建线粒体抗病毒信号蛋白真核表达载体.方法通过聚合酶链反应(PCR)的方法扩增获得线粒体抗病毒信号蛋白基因的完整序列,进而纯化回收后连接到pMDTM 18-T载体上,将pcDNA6/myc-HisA载体和带有目的片段的pMDTM 18-T载体同时进行双酶切,酶切产物过夜连接转化至大肠杆菌JM109,挑取阳性克隆pcDNA6/myc-HisA-MA VS扩增后,提取重组质粒;利用PCR和核酸测序验证线粒体抗病毒信号蛋白真核表达载体构建的正确性;应用Western blotting的方法检测融合蛋白的表达.结果线粒体抗病毒信号蛋白真核表达载体构建成功,并且该载体能够在OL细胞中表达融合蛋白.结论成功构建线粒体抗病毒信号蛋白真核表达载体,为进一步研究该蛋白的功能和作用机制奠定基础.
목적 구건선립체항병독신호단백진핵표체재체.방법 통과취합매련반응(PCR)적방법확증획득선립체항병독신호단백기인적완정서렬,진이순화회수후련접도pMDTM 18-T재체상,장pcDNA6/myc-HisA재체화대유목적편단적pMDTM 18-T재체동시진행쌍매절,매절산물과야련접전화지대장간균JM109,도취양성극륭pcDNA6/myc-HisA-MA VS확증후,제취중조질립;이용PCR화핵산측서험증선립체항병독신호단백진핵표체재체구건적정학성;응용Western blotting적방법검측융합단백적표체.결과 선립체항병독신호단백진핵표체재체구건성공,병차해재체능구재OL세포중표체융합단백.결론 성공구건선립체항병독신호단백진핵표체재체,위진일보연구해단백적공능화작용궤제전정기출.
Objective To construct the eukaryotic expression vector of mitochondrial antiviral signaling protein.Methods The target DNA fragments of mitochondrial antiviral signaling protein gene was obtained from PCR amplification,then the cDNA fragment was ligated into pMDTM18-T Vector.After double-enzyme digestion,the MAVS gene was transferred into the eukaryotic expression vector pcDNA6/myc-HisA.The constructs transformed into E.coil JM109 and positive clones were picked by pcDNA6/myc-HisA-MAVS PCR amplification.This construct was confirmed by PCR and DNA sequencing.Results The eukaryotic expression vector of mitochondrial antiviral signaling protein was constructed correctly.The recombinant vector was transfected into OL cells,and the expression of the recombinant protein was detected by western blotting.Conclusion The successfully constructed pcDNA6/myc-HisA MAVS plasmid is useful to study the gene's function and effects in cells.This study is for further study of the mechanism of gene function.