摘要
目的通过插入GS筛选系统和替换启动子改造载体以获得高效表达细胞系。方法将pIRES2-EGFP多克隆位点上的BamHⅠ位点突变,构建一个新的载体pIRES2-EGFP-B,通过AseⅠ和NheⅠ双酶切其上的巨细胞病毒(CMV)启动子,加入谷氨酰胺合成酶(GS)基因筛选标志,再通过PacⅠ和NheⅠ双酶切,插入人巨细胞病毒(hCMV)启动子,构建载体pHGS1.0。将目的基因分别插入载体pHGS1.0和pIRES2-EGFP中,通过比较重组蛋白TEM8(1-227)-VEGFR1domain2-IgG2(TV-IgG2)表达来分析新构建载体的优越性。结果成功插入GS筛选标志,并加入hCMV启动子,人免疫球蛋白定量试剂盒分析发现,重组蛋白表达量提高了5倍。结论通过GS系统筛选,得到高效表达目的蛋白载体系统,为后续的高效表达细胞系筛选奠定了基础。
Objective To obtain highly expressing cell lines by inserting the glutamine synthetase （GS） screening system and replacing the promoter of the vector.Methods The mutation of the point BamHⅠwas induced to build a new vector pIRES2-EGFP.The marker gene GS was inserted by AseⅠ and NheⅠ, and the promoter hCMV was replaced by PacⅠand NheⅠ.The new vector pHGS1.0 and the vector pIRES2-enhanced screen fluorescein protein（ EGFP）-B were inserted by the recombinant protein TEM8 （ 1-227 ）-VEGFR1 domain2-IgG2 （ TV-IgG2 ） gene to analyze the advantages of the expression.Results The glutamine synsthetase is successfully inserted, the human cytomegalovirus replaced, and recombinant protein is increased 5-fold by human immunoglobulin quantification kit.Conclusion The GS system is a highly protein expressing system.
出处
《军事医学》
CAS
CSCD
北大核心
2014年第10期807-810,共4页
Military Medical Sciences
基金
"重大新药创制"国家科技重大专项资助项目(2011ZX09102-001-30
2012ZX09102301-001)
