中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2011年
11期
767-771
,共5页
龚苗子%由江峰%崔湘林%郑杰
龔苗子%由江峰%崔湘林%鄭傑
공묘자%유강봉%최상림%정걸
肿瘤转移%基因,肿瘤抑制%绿色荧光蛋白质类%核仁组成区%细胞系,肿瘤
腫瘤轉移%基因,腫瘤抑製%綠色熒光蛋白質類%覈仁組成區%細胞繫,腫瘤
종류전이%기인,종류억제%록색형광단백질류%핵인조성구%세포계,종류
Neoplasm metastasis%Genes,tumor suppressor%Green fluorescent proteins%Nucleolus organizer region%Cell line,tumor
目的 鉴定肿瘤转移抑制相关基因TMSG-1蛋白中潜在的特异性定位信号序列并探索其亚细胞定位机制.方法 聚合酶链反应(PCR)扩增TMSG-1开放读码框全长及不同长度的截断片段,定向克隆于绿色荧光蛋白(GFP)表达质粒pEGFP-N1;各融合蛋白表达质粒转染人胚肾细胞系HEK293细胞；转染48 h后提取细胞总蛋白进行GFP的Western blot检测或用冷丙酮固定细胞后激光共聚焦显微镜观察融合蛋白的亚细胞定位.结果 GFP分别融合TMSG-1全长蛋白(aa1-380)及其截断片段T1(aa1-70)、T2(aa1-128)、T3(aa129-380)、T4(aa71-128)、T5(aa71-179)和T6(aa71-380),Western blot检测结果显示成功表达了各融合蛋白.激光共聚焦显微镜观察亚细胞定位显示融合蛋白T4(aa71-128)主要定位于细胞核内的核仁部位,融合蛋白T6(aa71-380)以细胞核内弥散分布为主,而TMSG-1全长融合蛋白及融合蛋白T1、T2、T3、T5则定位于胞质.进一步的序列缺失去除T4( aa71-128)羧基末端10个氨基酸得到截断片段T4Δ119-128,T4Δ119-128融合的GFP仍位于细胞核,但核仁内的绿色荧光信号明显减弱.结论 肿瘤转移抑制相关基因TMSG-1存在潜在的核仁定位信号,位于aa119-128(RRRRNQDRPS),这一发现为深入研究TMSG-1的亚细胞定位及相关功能奠定了基础.
目的 鑒定腫瘤轉移抑製相關基因TMSG-1蛋白中潛在的特異性定位信號序列併探索其亞細胞定位機製.方法 聚閤酶鏈反應(PCR)擴增TMSG-1開放讀碼框全長及不同長度的截斷片段,定嚮剋隆于綠色熒光蛋白(GFP)錶達質粒pEGFP-N1;各融閤蛋白錶達質粒轉染人胚腎細胞繫HEK293細胞；轉染48 h後提取細胞總蛋白進行GFP的Western blot檢測或用冷丙酮固定細胞後激光共聚焦顯微鏡觀察融閤蛋白的亞細胞定位.結果 GFP分彆融閤TMSG-1全長蛋白(aa1-380)及其截斷片段T1(aa1-70)、T2(aa1-128)、T3(aa129-380)、T4(aa71-128)、T5(aa71-179)和T6(aa71-380),Western blot檢測結果顯示成功錶達瞭各融閤蛋白.激光共聚焦顯微鏡觀察亞細胞定位顯示融閤蛋白T4(aa71-128)主要定位于細胞覈內的覈仁部位,融閤蛋白T6(aa71-380)以細胞覈內瀰散分佈為主,而TMSG-1全長融閤蛋白及融閤蛋白T1、T2、T3、T5則定位于胞質.進一步的序列缺失去除T4( aa71-128)羧基末耑10箇氨基痠得到截斷片段T4Δ119-128,T4Δ119-128融閤的GFP仍位于細胞覈,但覈仁內的綠色熒光信號明顯減弱.結論 腫瘤轉移抑製相關基因TMSG-1存在潛在的覈仁定位信號,位于aa119-128(RRRRNQDRPS),這一髮現為深入研究TMSG-1的亞細胞定位及相關功能奠定瞭基礎.
목적 감정종류전이억제상관기인TMSG-1단백중잠재적특이성정위신호서렬병탐색기아세포정위궤제.방법 취합매련반응(PCR)확증TMSG-1개방독마광전장급불동장도적절단편단,정향극륭우록색형광단백(GFP)표체질립pEGFP-N1;각융합단백표체질립전염인배신세포계HEK293세포；전염48 h후제취세포총단백진행GFP적Western blot검측혹용랭병동고정세포후격광공취초현미경관찰융합단백적아세포정위.결과 GFP분별융합TMSG-1전장단백(aa1-380)급기절단편단T1(aa1-70)、T2(aa1-128)、T3(aa129-380)、T4(aa71-128)、T5(aa71-179)화T6(aa71-380),Western blot검측결과현시성공표체료각융합단백.격광공취초현미경관찰아세포정위현시융합단백T4(aa71-128)주요정위우세포핵내적핵인부위,융합단백T6(aa71-380)이세포핵내미산분포위주,이TMSG-1전장융합단백급융합단백T1、T2、T3、T5칙정위우포질.진일보적서렬결실거제T4( aa71-128)최기말단10개안기산득도절단편단T4Δ119-128,T4Δ119-128융합적GFP잉위우세포핵,단핵인내적록색형광신호명현감약.결론 종류전이억제상관기인TMSG-1존재잠재적핵인정위신호,위우aa119-128(RRRRNQDRPS),저일발현위심입연구TMSG-1적아세포정위급상관공능전정료기출.
Objective To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein.Methods Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 ( HEK293 ) cells.The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope.Results GFP was fused with the native TMSG-1 ( aal-380 ) or different fragments including T1 (aa1-70),T2 (aa1-128),T3 (aa129-380),T4 (aa71-128),T5 (aa71-179) and T6 (aa71-380 ).Anti-GFP Western blotting showed that these fusion proteins were successfully expressed.Under laser confocal microscope,GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fusedwith fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aal-380) or fragment T1 (aal-70),T2 (aa1-128),T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm.Fragment T4Δ119-128 was generated from T4 with deletion of 10 amino acid of the C terminal.GFP fused with fragment T4Δ119-128 remained in the nucleus,but no longer in the nucleolus.Conclusions There is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1.This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.
