CAJ | 학술논문

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GDNF DNA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-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-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16 000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.
목적 재대장간균중표체구유생물활성적인효질세포원성신경영양인자(glial cell linederived neurotrophic factor,GDNF)단백.방법 종인효질류조직중제취총RNA,채용역전록취합매련반응(reverse transcription PCR,RT-PCR)방법확증출GDNF DNA편단,병장경쌍매절후적GDNF기인편단직접여표체재체pET28a+련접,구건중조표체재체pET28a+-GDNF,재장기전입대장간균BL21중진행이병기-β-D-류대반유당감유도표체;채용분자사층석법대표체산물진행순화;용십이완기류산납-취병희선알응효전영(SDS-PAGE)대기순도진행감정.결과 매절화측서증실,PCR확증출GDNF기인편단위GDNF성숙태편마서렬;표체산물적SDS-PAGE현시,상대분자질량16 000처유일신증단백대.결론 본연구소구건적원핵표체계통가표체인GDNF.
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 M.16 000protein band. Conclusion The prokaryotic expression system we constructed can express human GDNF.