摘要
人工合成表皮生长因子受体干扰序列(epithelial growth factor receptor interference,EGFRi),应用重叠延伸PCR(Overlapping PCR)技术将其与白细胞介素-24(IL-24)连接,并在其间引入一段柔软短肽(Gly4Ser3)。进行序列分析后,将融合基因EGFRi-IL-24重组到表达质粒pPIC9k中,Sal I酶切线性化重组质粒EGFRi-IL-24/pPIC9k,电击转化毕赤酵母GS115,经G418抗性和PCR筛选得到阳性重组菌株。重组菌株用甲醇进行诱导表达后,通过SDS-PAGE和MTT法分析鉴定蛋白表达产物。结果证实EGFRi-IL-24在毕赤酵母中获得了分泌性表达的活性蛋白,为进一步研究其生物学功能及临床应用奠定了基础。
The epithelial growth factor receptor interference（EGFRi） was obtained by synthetic primers.Overlapping PCR was used to produce EGFRi-IL-24 fusion gene,which is linked by Gly4Ser3.After sequence analysis,EGFRi-IL-24 was cloned into expression vector pPIC9k;EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115.Subsequently,positive clone was selected by G418 and PCR,and its phenotype was determined by SDS-PAGE and MTT assay.The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2010年第2期395-399,共5页
Journal of Biomedical Engineering
基金
天津科技大学自然科学基金资助项目(20060206)
关键词
表皮生长因子受体干扰序列
毕赤酵母
蛋白表达
Epithelial growth factor receptor interference（EGFRi）
Piehia postoris
Protein expression
