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

目的：构建人补体膜辅助调节蛋白（membrane cofactor protein，MCP）真核表达载体并利用壳聚糖（Chitosan）转染小鼠NIH 3T3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1065bp，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。

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

Objective:To construct the eukaryotic expression vector of human membrane cofactor protein(MCP) and transfect it into NIH 3T_3 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 pSecTaG_2/HygroB,and identified by restriction endonuclease's digestion and DNA sequencing.After the particles of pSecTaG_2/HygroB-MCP were encapsulated by chitosan,the NIH 3T_3 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 3T_3 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 3T_3 cells with chitosan-MCP nanoparticles,which may be very helpful for the transgenic liver study.