天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
6期
522-525
,共4页
高星杰%宋娟%葛林%付雪%孙晓明%张纬%何津岩%姚智%杨洁
高星傑%宋娟%葛林%付雪%孫曉明%張緯%何津巖%姚智%楊潔
고성걸%송연%갈림%부설%손효명%장위%하진암%요지%양길
核糖核蛋白类,核不均一%绿色荧光蛋白质类%膜融合蛋白质类%hnRNP A1蛋白%pEGFP-C1%融合蛋白%应激颗粒
覈糖覈蛋白類,覈不均一%綠色熒光蛋白質類%膜融閤蛋白質類%hnRNP A1蛋白%pEGFP-C1%融閤蛋白%應激顆粒
핵당핵단백류,핵불균일%록색형광단백질류%막융합단백질류%hnRNP A1단백%pEGFP-C1%융합단백%응격과립
heterogeneous-nuclear ribonucleoproteins%green fluorescent proteins%membrane fusion proteins%human hnRNP A1 protein%pEGFP-C1%fusion protein%stress granules
目的:构建包含有核不均一核糖核蛋白(hnRNP)A1蛋白编码区序列的真核表达质粒pEGFP-C1-hnRNP A1,并对增强型绿色荧光蛋白(EGFP)标记的hnRNP A1蛋白进行细胞应激共定位分析。方法提取HeLa细胞总RNA,以针对hnRNPA1-3′非翻译区的特异性片段为反转录引物,反转录出包含hnRNP A1编码区序列的cDNA,并以其为模板,降落PCR法扩增出带EcoRⅠ和BamHⅠ双酶切位点的目的基因,利用双酶切法分别酶切目的基因片段和线性pEGFP-C1,在T4-DNA连接酶的催化下将两者连接构建成pEGFP-C1-hnRNP A1重组质粒,然后将重组质粒转染入HeLa细胞内,以激光共聚焦荧光显微镜观察EGFP-hnRNP A1的荧光表达情况,Western印迹法检测EGFP与hnRNP A1的融合表达情况,最后进行细胞原位杂交及细胞免疫荧光检测在氧化应激状态下EGFP-hnRNP A1蛋白与poly(A)+mRNA(应激颗粒的标记成分)及DPC1a(加工体的标记蛋白)的应激共定位。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误,激光共聚焦荧光显微镜观察和Western印迹结果检测到绿色荧光融合蛋白的表达;EGFP-hnRNP A1蛋白与poly(A)+mRNA呈现共定位,但与DPC1a无共定位关系。结论重组pEGFP-C1-hnRNP A1质粒成功构建并表达,应激状态下EGFP标记的hnRNP A1参与应激颗粒的构成。
目的:構建包含有覈不均一覈糖覈蛋白(hnRNP)A1蛋白編碼區序列的真覈錶達質粒pEGFP-C1-hnRNP A1,併對增彊型綠色熒光蛋白(EGFP)標記的hnRNP A1蛋白進行細胞應激共定位分析。方法提取HeLa細胞總RNA,以針對hnRNPA1-3′非翻譯區的特異性片段為反轉錄引物,反轉錄齣包含hnRNP A1編碼區序列的cDNA,併以其為模闆,降落PCR法擴增齣帶EcoRⅠ和BamHⅠ雙酶切位點的目的基因,利用雙酶切法分彆酶切目的基因片段和線性pEGFP-C1,在T4-DNA連接酶的催化下將兩者連接構建成pEGFP-C1-hnRNP A1重組質粒,然後將重組質粒轉染入HeLa細胞內,以激光共聚焦熒光顯微鏡觀察EGFP-hnRNP A1的熒光錶達情況,Western印跡法檢測EGFP與hnRNP A1的融閤錶達情況,最後進行細胞原位雜交及細胞免疫熒光檢測在氧化應激狀態下EGFP-hnRNP A1蛋白與poly(A)+mRNA(應激顆粒的標記成分)及DPC1a(加工體的標記蛋白)的應激共定位。結果以單/雙酶切及基因測序法鑒定構建的重組質粒無誤,激光共聚焦熒光顯微鏡觀察和Western印跡結果檢測到綠色熒光融閤蛋白的錶達;EGFP-hnRNP A1蛋白與poly(A)+mRNA呈現共定位,但與DPC1a無共定位關繫。結論重組pEGFP-C1-hnRNP A1質粒成功構建併錶達,應激狀態下EGFP標記的hnRNP A1參與應激顆粒的構成。
목적:구건포함유핵불균일핵당핵단백(hnRNP)A1단백편마구서렬적진핵표체질립pEGFP-C1-hnRNP A1,병대증강형록색형광단백(EGFP)표기적hnRNP A1단백진행세포응격공정위분석。방법제취HeLa세포총RNA,이침대hnRNPA1-3′비번역구적특이성편단위반전록인물,반전록출포함hnRNP A1편마구서렬적cDNA,병이기위모판,강락PCR법확증출대EcoRⅠ화BamHⅠ쌍매절위점적목적기인,이용쌍매절법분별매절목적기인편단화선성pEGFP-C1,재T4-DNA련접매적최화하장량자련접구건성pEGFP-C1-hnRNP A1중조질립,연후장중조질립전염입HeLa세포내,이격광공취초형광현미경관찰EGFP-hnRNP A1적형광표체정황,Western인적법검측EGFP여hnRNP A1적융합표체정황,최후진행세포원위잡교급세포면역형광검측재양화응격상태하EGFP-hnRNP A1단백여poly(A)+mRNA(응격과립적표기성분)급DPC1a(가공체적표기단백)적응격공정위。결과이단/쌍매절급기인측서법감정구건적중조질립무오,격광공취초형광현미경관찰화Western인적결과검측도록색형광융합단백적표체;EGFP-hnRNP A1단백여poly(A)+mRNA정현공정위,단여DPC1a무공정위관계。결론중조pEGFP-C1-hnRNP A1질립성공구건병표체,응격상태하EGFP표기적hnRNP A1삼여응격과립적구성。
Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.