本文選題：表皮生長(zhǎng)因子受體　切入點(diǎn)：酪氨酸激酶結(jié)構(gòu)域　出處：《福建醫(yī)科大學(xué)》2006年碩士論文

【摘要】： 目的: 1.研究人表皮生長(zhǎng)因子受體(epidermal growth factor receptor, EGFR)胞內(nèi)酪氨酸激酶結(jié)構(gòu)域(tyrosine kinase domain,TKD)的原核表達(dá)及純化條件;2.研究在人腫瘤細(xì)胞中穩(wěn)定表達(dá)人表皮生長(zhǎng)因子受體胞內(nèi)酪氨酸激酶結(jié)構(gòu)域。 方法:1.構(gòu)建原核表達(dá)人EGFR胞內(nèi)酪氨酸激酶結(jié)構(gòu)域的重組質(zhì)粒pGEX/GST-EGFR-TKD,大腸桿菌表達(dá)目的融合蛋白GST-EGFR-TKD,經(jīng)尿素變性、梯度尿素透析復(fù)性、純化GST融合蛋白、腸激酶去除GST“標(biāo)簽”后獲得人EGFR胞內(nèi)酪氨酸激酶結(jié)構(gòu)域蛋白; 2.采用磷酸鈣共沉淀基因轉(zhuǎn)染技術(shù),將已構(gòu)建的pEF-BOS/GST-EGFR-TKD質(zhì)粒DNA與pBabe-puro質(zhì)粒DNA共同導(dǎo)入體外培養(yǎng)的人非小細(xì)胞肺癌細(xì)胞系H1299中,嘌呤霉素加壓篩選轉(zhuǎn)染細(xì)胞以獲得成功轉(zhuǎn)染的細(xì)胞,免疫熒光法檢測(cè)重組質(zhì)粒在H1299中的表達(dá)水平及定位。 結(jié)果: 1.限制性內(nèi)切酶分析和DNA測(cè)序表明已成功構(gòu)建重組質(zhì)粒pGEX/GST-EGFR-TKD,SDS-PAGE分析表明異丙基硫代-β-D半乳糖苷(IPTG)誘導(dǎo)下可表達(dá)融合蛋白GST-EGFR-TKD,但以不溶性包涵體的形式存在。經(jīng)尿素變性,梯度透析復(fù)性及腸激酶去除GST“標(biāo)簽”后可獲得重折疊的人EGFR胞內(nèi)酪氨酸激酶結(jié)構(gòu)域蛋白; 2.經(jīng)嘌呤霉素篩選, 8d后對(duì)照組細(xì)胞全部死亡。轉(zhuǎn)染組一些細(xì)胞存活形成單克隆集落,經(jīng)多次傳代培養(yǎng)后,熒光顯微鏡下觀察見多數(shù)集落的細(xì)胞胞質(zhì)內(nèi)有較強(qiáng)GST-EGFR-TKD表達(dá)。 結(jié)論: 1.在大腸桿菌中可成功表達(dá)人EGFR胞內(nèi)酪氨酸激酶結(jié)構(gòu)域蛋白;2.通過磷酸鈣共沉淀法可成功轉(zhuǎn)染H1299細(xì)胞,并獲得穩(wěn)定表達(dá)EGFR-TKD的人腫瘤細(xì)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

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本文編號(hào)：1713976