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大鼠热休克蛋白47真核表达载体的体外构建 被引量：2

Construction of Recombinant Plasmid pTracer-CMV-HSP47 in vitro and its Expression in NIH/3T3
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摘要 目的:体外构建大鼠热休克蛋白47(47kDa heat shock protein,HSP47)的荧光真核表达载体,并检测其在鼠胚胎成纤维细胞NIH/3T3中的表达。方法:应用RT-PCR方法从大鼠肝脏中分离、扩增目的片断,双酶切定向克隆到真核表达载体pTracer-CMV中,构成重组质粒pTracer-CMV-HSP47,测序验证。通过脂质体介导质粒瞬时转染NIH/3T3细胞,Western blotting和免疫细胞化学检测HSP47及Ⅰ型胶原蛋白(collagenⅠ)的表达变化。结果:通过RT-PCR获得1.3kb的cDNA片断,测序发现除个别碱基差异外,其余cDNA序列与基因库(Genebank)中序列一致,且编码的氨基酸序列完全一致。转染后经Western blotting和免疫细胞化学检测证实HSP47和collagenⅠ表达明显增强。结论:本研究成功构建了大鼠HSP47荧光真核表达载体pTracer-CMV-HSP47,并能在NIH/3T3细胞中表达,同时证实HSP47能促进collagenⅠ的表达。 Objective： To construct fluorescent recombinant plasmid pTracer-CMV-HSP47 in vitro and to evaluate its expression in NIH/3T3 cells. Methods： The target sequence of rat HSP47 cDNA was obtained and amplified from rat liver by RT-PCR. The cDNA segment was subcloned into a eukaryote plasmid pTracer-CMV by directed cloning after two restrictive endonucleases digestion. The pTracer-CMV-HSP47 was checked and verified by DNA sequence analysis. NIH! 313 cells were transiently transfected with pTracer-CMV-HSP47 by lipofectamine 2000 in vitro. Western blotting and immunocytochemical analysis were employed to detect the expression changes of HSP47 and collagen Ⅰ. Results： The 1.3kb cDNA fragment was obtained by RT-PCR and subcloned into pTracer-CMV. The recombinant plasmid pTracerCMV-HSP47 was subject to sequence analysis which indicated two single nucleotides appeared different from the rat HSP47 sequence provided by Genebank. The amino acid sequence translated from the cDNA was matched perfectly with that of rat HSP47. Being transfected by Lipofectamine 2000, the expressions of HSP47 and collagen Ⅰ in NIH/3T3 were intensified. Conclusion： Recombinant fluorescent plasmid pTraeer-CMV-HSP47 was constructed in vitro and expressed successfully in NIH/3T3. HSP47 was also proved capable to enhance expression of collagenⅠ.
作者 程杰 王佐林
机构地区 同济大学附属口腔医院口腔颌面外科
出处 《口腔颌面外科杂志》 CAS 2007年第1期6-10,共5页 Journal of Oral and Maxillofacial Surgery
基金 国家自然科学基金面上项目(30471905) 上海市科委科研计划项目(034119840 05PJ14097)
关键词 热休克蛋白47 基因克隆 基因转染 质粒 47 kDa heat shock protein （HSP47） gene clone gene transfection plasmid
分类号 R346 [医药卫生—基础医学]
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参考文献14

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二级参考文献46

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共引文献5

同被引文献33

  • 1金啸,王佐林.热休克蛋白47在糖尿病大鼠皮肤创口愈合过程中的表达[J].口腔颌面外科杂志,2007,17(1):11-16. 被引量：1
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口腔颌面外科杂志
2007年 第1期

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