人胶质细胞源性神经营养因子基因片段克隆及其在大肠杆菌中的表达  

Gene cloning of human glial cell line-derived neurotrophic factor and its expression in E. coli

作　　者：刘亚珍[1] 施雨露[2] 王友联[3] 刘永茂[2] 万全[4] 

机构地区：[1]吉林大学第一医院药剂科,长春130021 [2]吉林大学白求恩医学院实验中心,长春130021 [3]白求恩医科大学制药厂,长春130021 [4]吉林大学生命科学学院08级生物技术系,长春130021

出　　处：《国际生物制品学杂志》2011年第1期1-4,共4页International Journal of Biologicals

基　　金：基金项目：吉林省科技发展计划（20050403-2）

摘　　要：目的在大肠杆菌中表达具有生物活性的人胶质细胞源性神经营养因子（glial cell linederived neurotrophic factor, GDNF）蛋白。方法从人胶质瘤组织中提取总RNA，采用逆转录聚合酶链反应（reverse transcription PCR，RT—PCR）方法扩增出GDNFDNA片段，并将经双酶切后的GDNF基因片段直接与表达载体pET28a＋连接，构建重组表达载体pET28a＋GDNF，再将其转入大肠杆菌BL21中进行异丙基-β—D一硫代半乳糖苷诱导表达；采用分子筛层析法对表达产物进行纯化；用十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）对其纯度进行鉴定。结果酶切和测序证实，PCR扩增出GDNF基因片段为GDNF成熟肽编码序列；表达产物的SDS—PAGE显示，相对分子质量16000处有一新增蛋白带。结论本研究所构建的原核表达系统可表达人GDNF。Objective To express human glial cell line-derived neurotrophic factor （GDNF） in E. coli （BL21）. Methods Gene segment of GDNF was amplified using the genomic DNA from human glioma as template by polymerase chain reaction, and was directly ligated with vector pET28a＋. The recombinant expression vector pET28a＋ -GDNF was transformed into E. coli BL21 （DE3） and induced with isopropyl β-D- thiogalactoside. The expression product was identified by SDS-PAGE after gel chromatography isolation. Results Gene segment of pET28a＋ -GDNF was identified as the sequence of encoding mature peptide of GDNF by restriction analysis and DNA sequencing. SDS-PAGE revealed that the purified product had a Mt 16 000 protein band. Conclusion The prokaryotie expression system we constructed can express human GDNF.

关 键 词：神经生长因子类 基因表达 逆转录聚合酶链反应 

分 类 号：R[医药卫生]