天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
7期
625-629
,共5页
高星杰%何津岩%葛林%张毅%付雪%尹洁%张纬%史雪彬%苏征%姚智%杨洁
高星傑%何津巖%葛林%張毅%付雪%尹潔%張緯%史雪彬%囌徵%姚智%楊潔
고성걸%하진암%갈림%장의%부설%윤길%장위%사설빈%소정%요지%양길
重组融合蛋白质类%应激%质粒%基因表达%SND1%AUG%应激颗粒
重組融閤蛋白質類%應激%質粒%基因錶達%SND1%AUG%應激顆粒
중조융합단백질류%응격%질립%기인표체%SND1%AUG%응격과립
recombinant fusion proteins%stress%plasmids%gene expression%SND1%AUG%stress granules
目的：针对人SND1基因2个蛋白翻译起始密码子AUG构建真核表达质粒pCMV-N-Flag-SND1-No1/2，并分析2个AUG在SND1应激颗粒形成中的作用。方法以SND1全长转录本为模板，PCR法扩增含BamHⅠ和EcoRⅠ酶切位点的目的基因SND1-No1/2，双酶切法分别酶切目的基因片段和线性pCMV-N-Flag，以T4-DNA连接酶将两者连接成pCMV-N-Flag-SND1-No1/2重组质粒，然后将构建的重组质粒转染入HeLa细胞内，以Western印迹法检测Flag标签（DYKDDDDK）与SND1-No1/2的融合表达，最后以细胞免疫荧光实验检测在氧化应激状态下Flag-SND1-No1/2融合蛋白与内源性SND1应激颗粒的胞内共定位情况。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误，Western印迹结果检测到融合蛋白Flag-SND1-No1/2的表达；细胞免疫荧光结果显示Flag-SND1-No1/2均可与内源性SND1应激颗粒共定位。结论重组pCMV-N-Flag-SND1-No1/2质粒构建成功，SND1基因第1个AUG的缺失并不影响SND1应激颗粒的形成。
目的：針對人SND1基因2箇蛋白翻譯起始密碼子AUG構建真覈錶達質粒pCMV-N-Flag-SND1-No1/2，併分析2箇AUG在SND1應激顆粒形成中的作用。方法以SND1全長轉錄本為模闆，PCR法擴增含BamHⅠ和EcoRⅠ酶切位點的目的基因SND1-No1/2，雙酶切法分彆酶切目的基因片段和線性pCMV-N-Flag，以T4-DNA連接酶將兩者連接成pCMV-N-Flag-SND1-No1/2重組質粒，然後將構建的重組質粒轉染入HeLa細胞內，以Western印跡法檢測Flag標籤（DYKDDDDK）與SND1-No1/2的融閤錶達，最後以細胞免疫熒光實驗檢測在氧化應激狀態下Flag-SND1-No1/2融閤蛋白與內源性SND1應激顆粒的胞內共定位情況。結果以單/雙酶切及基因測序法鑒定構建的重組質粒無誤，Western印跡結果檢測到融閤蛋白Flag-SND1-No1/2的錶達；細胞免疫熒光結果顯示Flag-SND1-No1/2均可與內源性SND1應激顆粒共定位。結論重組pCMV-N-Flag-SND1-No1/2質粒構建成功，SND1基因第1箇AUG的缺失併不影響SND1應激顆粒的形成。
목적：침대인SND1기인2개단백번역기시밀마자AUG구건진핵표체질립pCMV-N-Flag-SND1-No1/2，병분석2개AUG재SND1응격과립형성중적작용。방법이SND1전장전록본위모판，PCR법확증함BamHⅠ화EcoRⅠ매절위점적목적기인SND1-No1/2，쌍매절법분별매절목적기인편단화선성pCMV-N-Flag，이T4-DNA련접매장량자련접성pCMV-N-Flag-SND1-No1/2중조질립，연후장구건적중조질립전염입HeLa세포내，이Western인적법검측Flag표첨（DYKDDDDK）여SND1-No1/2적융합표체，최후이세포면역형광실험검측재양화응격상태하Flag-SND1-No1/2융합단백여내원성SND1응격과립적포내공정위정황。결과이단/쌍매절급기인측서법감정구건적중조질립무오，Western인적결과검측도융합단백Flag-SND1-No1/2적표체；세포면역형광결과현시Flag-SND1-No1/2균가여내원성SND1응격과립공정위。결론중조pCMV-N-Flag-SND1-No1/2질립구건성공，SND1기인제1개AUG적결실병불영향SND1응격과립적형성。
Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1(from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co-localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to af-fect the formation of SND1 containing stress granules.
