河南医科大学学报2001，Vol.36Issue(3)：267-271,5.
四环素抗性表型筛选外源基因高效表达克隆
Screening of high expression clone of heterologous gene by using tetracycline resistance genotype
摘要
Abstract
Aim：To screen high expressed clone from random codon of heterologous gene using tetracycline resistance (TetR) genotype. Methods: TetR gene was cloned into pBV220 to construct pBV223. Random primer codon bank of heterologous gene (for example hGM-CSF cDNA) was synthesized under the condition of changing 7 bases with the original amino acid sequence maintained. PCR amplified the bank of hGM-CSF gene, then the gene bank was cloned to site of EcoR Iand BamHI of pBV223 to construct pBV223/hGM-CSF library. TetR gene followed hGM-CSF down-stream and there were only six bases between the two genes. hGM-CSF cDNA expression drived TetR gene expression, and their expression levels were correlative. pBV223/hGM-CSF library was transferred into E.coli. DH5α and cultured in tetracycline culture media with different concentrations: 20mg/L, 40mg/L, 50mg/L and 60mg/L. Results: High experessed pBV223/hGM-CSF clone was found in high concentration tetracycline culture plate.The sequencing result demonstrated in 5′-terminal sequence of the hGM-CSF cDNA,7 bases changed when compared with the original one. The expression level of hGM-CSF in E.coli. DH5α was proximately 18% of the whole bacterial protein and enhanced about 80% in comparison with that of the original one. Conclussion: We could screen high expressed clone of heterologous gene using tetracycline resistance screening system.关键词
四环素抗性/hGM-CSF/克隆/筛选分类
生物科学引用本文复制引用
李玉富,孙声桃,李继昌,李福胜,张智清..四环素抗性表型筛选外源基因高效表达克隆[J].河南医科大学学报,2001,36(3):267-271,5.基金项目
国家“863”高科技计划资助项目 83-102-11-09 （）
