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

作　　者：李玉富[1] 孙声桃[2] 李继昌[3] 李福胜[4] 张智清[4] 

机构地区：[1]河南省肿瘤医院生物治疗中心,郑州450008 [2]河南省眼科研究所,郑州450003 [3]河南医科大学第一附属院内科,郑州450052 [4]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052

出　　处：《河南医科大学学报》2001年第3期267-271,共5页Journal of Henan Medical University

基　　金：国家"8 63"高科技计划资助项目! 83 10 2 11 0 9

摘　　要：目的 :应用四环素抗性表型从外源基因的cDNA文库中筛选高效表达克隆。方法 :将四环素抗性基因(TetR)克隆到载体pBV2 2 0 中构建筛选载体pBV2 2 3 ;根据氨基酸密码子的简并性 ,在保持原有氨基酸序列不变的情况下 ,人工合成外源基因的cDNA 5′端简并引物库 ,以hGM CSF基因为例进行筛选。把hGM CSFcDNA简并文库克隆到pBV2 2 3 中的TetR 上游位置 ,构建pBV2 2 3 /hGM CSF克隆文库 ,导入大肠杆菌DH5α中 ,构建转基因的细菌文库 ,在不同质量浓度 (2 0mg/L ,40mg/L ,5 0mg/L ,6 0mg/L)的四环素培养基中培养。结果 :从高浓度四环素培养基中获得高效表达的pBV2 2 3 /hGM CSF克隆 ,测序结果显示该克隆的hGM CSFcDNA 5′端序列与天然序列比较有 7个碱基发生突变 ,且符合实验设计的要求。通过基因重组 ,构建成最终的高效表达克隆pBV2 2 0 /hGM CSF ,其表达hGM CSF的量占细菌总量的 18% ,较原表达水平提高 80 %。结论 :可通过四环素抗性筛选系统筛选外源基因的高效表达克隆。Aim:To screen high expressed clone from random codon of heterologous gene using tetracycline resistance (Tet R) genotype. Methods: Tet R gene was cloned into p BV220 to construct p BV223 . 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 p BV223 to construct p BV223 /hGM CSF library. Tet R gene followed hGM CSF down stream and there were only six bases between the two genes. hGM CSF cDNA expression drived Tet R gene expression, and their expression levels were correlative. p BV223 /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 p BV223 /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GM-CSF 克隆 筛选 CDNA文库 外源基因 

分 类 号：Q785[生物学—分子生物学]