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构建人补体膜辅助调节蛋白真核表达载体并通过壳聚糖介导转染NIH 3T3细胞

Construction of the eukaryotic expression vector of human membrane cofactor protein and transfection NIH 3T3 cells with chitosan nanoparticles

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【作者】 乔庆陈勇窦科峰张静张传山

【Author】 QIAO Qing,CHEN Yong,DOU Ke-feng,et al.(Department of General Surgery,Tangdu Hospital,Fourth(Military) Medical University,710038,China)

【机构】 第四军医大学唐都医院普通外科第四军医大学西京医院肝胆外科第四军医大学西京医院病理科第四军医大学西京医院病理科 陕西西安710038陕西西安710038

【摘要】 目的:构建人补体膜辅助调节蛋白(m embrane cofactor prote in,MCP)真核表达载体并利用壳聚糖(Ch itosan)转染小鼠NIH3T3细胞。方法:应用PCR方法,从MCP-pGEM-T Easy Vector克隆相关的DNA序列,插入到具有相应酶切位点的真核表达载体pSecTag2/HygroB中,构建pSecTag2/HygroB-MCP真核表达质粒,并进行酶切鉴定及序列测定;利用壳聚糖包裹质粒,转染小鼠NIH 3T3细胞,并对转染细胞进行抗MCP免疫组织化学染色。结果:MCP基因大小为1 065 bp,与Genebank中记载的人MCP cDNA序列结果基本一致;抗MCP免疫组织化学染色显示转染细胞胞浆弥漫阳性。结论:成功构建了人MCP真核表达载体并通过壳聚糖介导转染NIH3T3细胞,为进一步探讨及研究转基因肝脏奠定了基础。

【Abstract】 Objective:To construct the eukaryotic expression vector of human membrane cofactor protein(MCP) and transfect it into NIH 3T3 cells with chitosan nanoparticles.Methods: The human MCP fragments were(obtained) by PCR from MCP-pGEM-T Easy Vector,cloned into the eukaryotic expression vector pSecTaG2/HygroB,and identified by restriction endonuclease’s digestion and DNA sequencing.After the particles of pSecTaG2/HygroB-MCP were encapsulated by chitosan,the NIH 3T3 cells were transfected by chitosan-MCP nanoparticles and MCP expression was detected by immunohistochemical stain.Results:The length of MCP fragment was 1 065 bp.Its(sequence) was approximately as same as MCP cDNA in Genbank.After having been transfected by chitosan-MCP nanoparticles for 24 hours,the NIH 3T3 cells showed diffusely positive in cytoplasms by anti-MCP(immunohistochemical) stain.Conclusion:We have successfully constructed the eukaryotic expression vector of(human)MCP and transfected it into NIH 3T3 cells with chitosan-MCP nanoparticles,which may be very helpful for the transgenic liver study.

【基金】 国家自然科学基金资助项目(30070210)
  • 【文献出处】 西北国防医学杂志 ,Medical Journal of National Defending Forces in Northwest China , 编辑部邮箱 ,2006年02期
  • 【分类号】R392
  • 【下载频次】46
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