摘要
目的:构建磷脂酰肌醇蛋白聚糖3(GPC3)真核表达载体pEGFP-GPC3,并在小鼠树突状细胞(DC)表达。方法:将质粒pCMV-SPORT6-GPC3和载体pEGFP-N1分别进行双酶切获得目的基因GPC3片段和空载体,回收纯化后用T4连接酶将两者连接并转化E.coli DH5α菌株,用EcoR I、BamH I双酶切鉴定后通过脂质体法转染到DC中,然后进行荧光检测和Western blot分析。结果:构建的真核表达载体pEGFP-GPC3,转染DC后,荧光显微镜下可见转染的DC中有EGFP-GPC3融合蛋白的表达,Western blot分析发现有相对分子质量(Mr)大小约为67 000的蛋白条带。结论:成功地构建了真核表达载体pEGFP-GPC3并在转染DC后能在其中表达,为进一步研究GPC3基因的功能提供实验基础。
AIM： To construct and expression of eukaryotic expression vector of glypican-3 （GPC3） pEGFP- GPC3 in mouse dendritic ceils （DCs）. METHODS： The target gene fragment of GPC3 and empty vector were obtained from plasmid pCMV-SPORT6-GPC3 and vector pEGFP-NI digested by double enzymes. GPC3 and empty vector were linked by ligase T4, and then transfected into E. coli DHSa. Identification with EcoR I, BamH I digestion, the recombinant eukaryotic expression vector pEGFP-GPC3 was transfected into DCs by LipofectamineTM2000. Fluores- cent microscopy and Western blot were used to detected the expression of GPC3. RESULTS： The expression of EGFP- GPC3 fusion protein was detected in DCs, and GPC3 fusion protein of 67 kDa was detected by Western blotting. CON- CLUSION： The eukaryotic expression vector pEGFP-of GPC3 was constructed succesfully and transfected effectively into DCs.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第5期462-465,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
广西医疗卫生重点科研课题(重200611)
广西科学研究与技术开发计划项目(桂科攻0719006-2-5)
广西科学基金(桂科自0728196)
