吴周环, 颜瑞巧, 刘建云, 等.核转运抑制肽表达载体的构建及对HeLa细胞增殖、迁移的影响.四川大学学报(医学版),2017,48(6):824-827
核转运抑制肽表达载体的构建及对HeLa细胞增殖、迁移的影响
Construction of Vectors Expressing Inhibiting Peptides for Nuclear Import and Its Effect on Growth and Migration of HeLa Cell
  
中文关键词:  核转运红色荧光蛋白增殖迁移
英文关键词:Nuclear importRed fluorescent proteinProliferationMigration
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中文摘要:
      目的 构建核转运抑制肽与红色荧光蛋白(RFP)的融合表达载体,探讨该融合蛋白在HeLa细胞中的表达、定位以及对细胞增殖、迁移的影响。方法 分别合成编码两种核转运抑制肽Bimax1和Bimax2的多核苷酸,退火后插入红色荧光蛋白表达载体pDs-Red-C1;质粒转化大肠杆菌DH-5α,挑取阳性菌进行DNA测序;利用脂质体转染法将重组载体pDs-Red-Bimax1、pDs-Red-Bimax2及空白载体转入HeLa细胞中,荧光显微镜观察红色荧光的表达与定位;MTT实验和细胞迁移实验检测融合蛋白表达对细胞增殖、迁移的影响。结果 DNA测序显示,两种核转运抑制肽Bimax1和Bimax2成功插入红色荧光蛋白表达载体;转染真核细胞后Bimax1和Bimax2引导红色荧光蛋白在细胞核表达;同时,融合蛋白RFP-Bimax1和RFP-Bimax2对HeLa细胞的增殖、迁移有抑制作用。结论 成功构建核转运特异性抑制肽与红色荧光蛋白融合表达的载体,融合蛋白在细胞核局限表达并能抑制肿瘤细胞的增殖、迁移。
英文摘要:
      Objective To construct the expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import (Bimax), and explore the location of Bimax and its potential effects on cell proliferation and migration in HeLa cells. Methods Two kinds of polynucleotide encoding inhibiting peptides for nuclear import were synthesis respectively and subsequently annealed for inserting into vector pDs-Red-C1. The recombinant plasmids were transfected into competent bacterial DH-5α. After transfection, the positive bacteria were picked up for DNA sequencing. The recombinant plasmids pDs-Red-Bimax2, pDs-Red-Bimax1 and negative plasmid pDs-Red-C1 were transfected into HeLa cells respectively according to Lipofectamine2000 protocol. After transfection, the expression and location of red fluorescent protein were observed with fluorescence microscope. Furthermore, MTT assay and cell-migration assay were used to detect the proliferation and migration of Bimax transducted cells. ResultsDNA sequencing showed that the polynucleotides encoding Bimax1 or Bimax2 were inserted into pDs-Red-C1 vector successfully. After transfected into HeLa cells, the inhibiting peptide induced red fluorescent protein locating in nuclear. Furthermore, either the fusion protein RFP-Bimax1 or RFP-Bimax2 can suppress the proliferation and migration of HeLa cells. Conclusion The expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import were successfully constructed. In addition, the fusion proteins were expressed and located in nuclear and suppressed the proliferation and migration of tumor cells.
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