表皮生长因子受体胞内酪氨酸激酶结构域的原核及真核表达研究

发布时间：2018-04-05 08:30

本文选题：表皮生长因子受体　切入点：酪氨酸激酶结构域　出处：《福建医科大学》2006年硕士论文

【摘要】： 目的: 1.研究人表皮生长因子受体(epidermal growth factor receptor, EGFR)胞内酪氨酸激酶结构域(tyrosine kinase domain,TKD)的原核表达及纯化条件;2.研究在人肿瘤细胞中稳定表达人表皮生长因子受体胞内酪氨酸激酶结构域。方法:1.构建原核表达人EGFR胞内酪氨酸激酶结构域的重组质粒pGEX/GST-EGFR-TKD,大肠杆菌表达目的融合蛋白GST-EGFR-TKD,经尿素变性、梯度尿素透析复性、纯化GST融合蛋白、肠激酶去除GST“标签”后获得人EGFR胞内酪氨酸激酶结构域蛋白; 2.采用磷酸钙共沉淀基因转染技术,将已构建的pEF-BOS/GST-EGFR-TKD质粒DNA与pBabe-puro质粒DNA共同导入体外培养的人非小细胞肺癌细胞系H1299中,嘌呤霉素加压筛选转染细胞以获得成功转染的细胞,免疫荧光法检测重组质粒在H1299中的表达水平及定位。结果: 1.限制性内切酶分析和DNA测序表明已成功构建重组质粒pGEX/GST-EGFR-TKD,SDS-PAGE分析表明异丙基硫代-β-D半乳糖苷(IPTG)诱导下可表达融合蛋白GST-EGFR-TKD,但以不溶性包涵体的形式存在。经尿素变性,梯度透析复性及肠激酶去除GST“标签”后可获得重折叠的人EGFR胞内酪氨酸激酶结构域蛋白; 2.经嘌呤霉素筛选, 8d后对照组细胞全部死亡。转染组一些细胞存活形成单克隆集落,经多次传代培养后,荧光显微镜下观察见多数集落的细胞胞质内有较强GST-EGFR-TKD表达。结论: 1.在大肠杆菌中可成功表达人EGFR胞内酪氨酸激酶结构域蛋白;2.通过磷酸钙共沉淀法可成功转染H1299细胞,并获得稳定表达EGFR-TKD的人肿瘤细胞。
[Abstract]:Objective: 1.To study the prokaryotic expression and purification conditions of human epidermal growth factor receptor (EGFR) tyrosine kinase domain.To study the stable expression of human epidermal growth factor receptor tyrosine kinase domain in human tumor cells.Method 1: 1.The recombinant plasmid pGEX / GST-EGFR-TKD was constructed to express the tyrosine kinase domain in human EGFR cells. The fusion protein GST-EGFR-TKD was expressed in E. coli. The fusion protein was purified by urea denaturation and gradient urea dialysis renaturation.The tyrosine kinase domain protein of human EGFR was obtained by removing the GST label.Using calcium phosphate coprecipitation gene transfection technique, the constructed pEF-BOS/GST-EGFR-TKD plasmid DNA and pBabe-puro plasmid DNA were co-transfected into human non-small cell lung cancer cell line H1299 in vitro. Purine mycin was pressurized to screen the transfected cells in order to obtain the successfully transfected cells.The expression level and localization of recombinant plasmid in H 1299 were detected by immunofluorescence assay.Results: 1.Restriction endonuclease analysis and DNA sequencing showed that the recombinant plasmid pGEX / GST-EGFR-TKDS-PAGE was successfully constructed. The results showed that the fusion protein GST-EGFR-TKDwas induced by isopropylthiothio- 尾 -D galactoside (IPTGG), but existed as an insoluble inclusion body.After urea denaturation, gradient dialysis renaturation and enterokinase removal of GST label, refolded tyrosine kinase domain proteins in human EGFR cells were obtained. 2.All the cells in the control group died after 8 days after purine mycin screening.In the transfection group, some cells survived and formed a monoclonal colony. After repeated passage culture, strong GST-EGFR-TKD expression was observed in the cytoplasm of the most colony cells under fluorescence microscope.Conclusion: 1.Human EGFR tyrosine kinase domain protein 2 was successfully expressed in E. coli.Human tumor cells expressing EGFR-TKD stably were successfully transfected into H1299 cells by calcium superphosphate coprecipitation.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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