【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。更多还原

【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.更多还原