中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2009年
2期
126-128
,共3页
瘢痕疙瘩%载体蛋白质类%蛋白质相互作用域和基序%分泌性卷曲相关蛋白2%骨母细胞特异性因子2
瘢痕疙瘩%載體蛋白質類%蛋白質相互作用域和基序%分泌性捲麯相關蛋白2%骨母細胞特異性因子2
반흔흘탑%재체단백질류%단백질상호작용역화기서%분비성권곡상관단백2%골모세포특이성인자2
Keloid%Carrier proteins%Protein interaction domains and motifs%Secreted friz-zled-related protein 2%Osteoblast-specific factor 2
目的 验证分泌性卷曲相关蛋白2(SFRP2)与骨母细胞特异性因子2(OSF-2)的相互作用.方法 构建能在哺乳动物细胞中表达带人膜联蛋白(HA)标签的OSF-2融合蛋白重组载体pCMV-HA-OSF-2,经酶切鉴定确认后,与Myc-SFRP2重组真核表达载体pCMV-HA-SFRP2单独或共转染人HK293细胞,利用免疫共沉淀技术及蛋白质印迹法验证OSF-2与SFRP2的相互作用.结果 重组载体经酶切电泳后,显示近800 bp处的条带为酶切后的目的 片段SFRP2编码基因,近2500 bp处的条带为酶切后目的 基因OSF-2.表明pCMV-Myc-SFRP2和pCMV-HA-OSF-2均构建成功.HK293细胞单独转染pCMV-Myc-SFRP2或pCMV-HA-OSF-2后,未检测到HA-OSF-2的表达;当pCMV-Myc-SFRP2和pCMV-HA-OSF-2共转染后,经Myc抗体进行免疫沉淀可见HA-OSF-2表达.结论 HA-OSF-2的重组载体能在哺乳动物细胞中表达,HA-OSF-2与SFRP2间存在相互作用.
目的 驗證分泌性捲麯相關蛋白2(SFRP2)與骨母細胞特異性因子2(OSF-2)的相互作用.方法 構建能在哺乳動物細胞中錶達帶人膜聯蛋白(HA)標籤的OSF-2融閤蛋白重組載體pCMV-HA-OSF-2,經酶切鑒定確認後,與Myc-SFRP2重組真覈錶達載體pCMV-HA-SFRP2單獨或共轉染人HK293細胞,利用免疫共沉澱技術及蛋白質印跡法驗證OSF-2與SFRP2的相互作用.結果 重組載體經酶切電泳後,顯示近800 bp處的條帶為酶切後的目的 片段SFRP2編碼基因,近2500 bp處的條帶為酶切後目的 基因OSF-2.錶明pCMV-Myc-SFRP2和pCMV-HA-OSF-2均構建成功.HK293細胞單獨轉染pCMV-Myc-SFRP2或pCMV-HA-OSF-2後,未檢測到HA-OSF-2的錶達;噹pCMV-Myc-SFRP2和pCMV-HA-OSF-2共轉染後,經Myc抗體進行免疫沉澱可見HA-OSF-2錶達.結論 HA-OSF-2的重組載體能在哺乳動物細胞中錶達,HA-OSF-2與SFRP2間存在相互作用.
목적 험증분비성권곡상관단백2(SFRP2)여골모세포특이성인자2(OSF-2)적상호작용.방법 구건능재포유동물세포중표체대인막련단백(HA)표첨적OSF-2융합단백중조재체pCMV-HA-OSF-2,경매절감정학인후,여Myc-SFRP2중조진핵표체재체pCMV-HA-SFRP2단독혹공전염인HK293세포,이용면역공침정기술급단백질인적법험증OSF-2여SFRP2적상호작용.결과 중조재체경매절전영후,현시근800 bp처적조대위매절후적목적 편단SFRP2편마기인,근2500 bp처적조대위매절후목적 기인OSF-2.표명pCMV-Myc-SFRP2화pCMV-HA-OSF-2균구건성공.HK293세포단독전염pCMV-Myc-SFRP2혹pCMV-HA-OSF-2후,미검측도HA-OSF-2적표체;당pCMV-Myc-SFRP2화pCMV-HA-OSF-2공전염후,경Myc항체진행면역침정가견HA-OSF-2표체.결론 HA-OSF-2적중조재체능재포유동물세포중표체,HA-OSF-2여SFRP2간존재상호작용.
Objective To verify the interaction between secreted frizzled-related protein 2(SFRP2) and osteoblast-specific factor 2 (OSF-2). Methods HA-tagged OSF-2 fusion protein recombinant vector pCMV-HA-OSF-2, which could express in mammal cells was constructed, then identified by enzyme-cutting and transfected into human kidney 293 (HK293) cells with or without Mye-SFRP2 recombinant eukaryotic expression vector pCMV-HA-SFBP2. The interaction between SFRP2 and OSF-2 was detected through co-immunoprecipitation and Western blotting. Results In electrophoresis bath, target fragment of SFRP2 coding gene with 800 bp and target gene OSF-2 with 2500 bp could be seen respectively after enzyme-cut-ting, which showed that pCMV-Mye-SFRP2 and pCMV-HA-OSF-2 were constructed successfully. No HA-OSF-2 expression was detected after pCMV-Myc-SFRP'2 or pCMV-HA-OSF-2 transfection. Whereas, HA-OSF-2 expressed by Mye antibody immunoprecipitation after pCMV-Myc-SFRP2 and pCMV-HA-OSF-2 co-transfection. Conclusions HA-OSF-2 recombinant vector can express in mammal cells. Interaction exists between HA-OSF-2 and SFRP2.
