磷酸化Akt1(Thr308)位点突变真核表达载体的构建及其生物学功能研究  

Cloning and Functional Characterization of Akt1(Thr308)Site Mutation

作　　者：熊加秀 丁丽华[2] 刘文忠 张亚楠[2] 陈志达[4] 罗晓丽[2] 贾小萌 陈滢洁 叶棋浓[2] 卫勃[4] 

机构地区：[1]内蒙古医科大学基础医学院,内蒙古呼和浩特010059 [2]军事医学科学院生物工程研究所,北京100850 [3]神华神东总医院,陕西榆林719315 [4]解放军总医院普通外科,北京100853

出　　处：《生物技术通讯》2016年第1期1-6,共6页Letters in Biotechnology

基　　金：国家自然科学基金(81101883,81272231,31470773);北京市科技新星计划(2009A38,2011112)

摘　　要：目的:构建磷酸化Akt1(Thr308)位点突变真核表达载体,并瞬时转染胃癌耐药细胞,对其生物学功能进行初步检测。方法:以带有pc DNA3.0-Flag标签的Akt1质粒为模板,采用重组PCR技术扩增得到Akt1(T308A)(苏氨酸突变成丙氨酸)、Akt1(T308E)(苏氨酸突变成谷氨酸)位点突变编码区序列,将其插入pc DNA3.0-Flag载体,双酶切和测序验证后瞬时转染人胚肾293T细胞,Western印迹检测其表达情况;将突变质粒与空载体分别转染人胃癌耐药细胞BGC-823/c DDP,通过Western印迹和实时定量PCR检测Notch1蛋白和m RNA水平的变化,通过CCK-8法检测对耐药细胞生长曲线的影响。结果:双酶切和测序结果表明Akt1(T308A)、Akt1(T308E)的真核表达质粒构建成功,转染293T细胞后获得表达;转染人胃癌耐药细胞BGC-823/c DDP后,利用实时定量PCR和Western印迹证明去磷酸化Akt1(T308A)可抑制Notch1的转录和蛋白水平,模拟持续磷酸化Akt1(T308E)升高Notch1的转录和蛋白表达;细胞生长曲线结果显示,转染Akt1(T308A)较空载体耐药细胞生长慢,而Akt1(T308E)促进耐药细胞生长。结论:PI3K/Akt与Notch1信号通路的激活在胃癌耐药的发生、发展过程中发挥重要作用。Objective： To construct Akt（Thr308） site mutations eukaryotic expression vectors and detect its func- tion in drug-resistance cells. Methods： Aktl（T308A） and Aktl （T308E） were amplified from peDNA3.0-Flag- Aktl by recombinant PCR and were inserted into pcDNA3.0-Flag vector. The recombinant plasmids were identi- fied by enzyme digestion and sequencing, and were transfeeted into HEK293T cells and detected by Western blot. qRT-PCR and Western blot were used to detect the effect of Aktl（T308A） and Akt1（T308E） on Notehl mRNA and protein expression. CCK-8 assay was performed to investigate the effect of the site mutation on gastric cancer of drug-resistance cell proliferation. Results： The Aktl （T308A） and Aktl （T308E） eukaryotie expression vectors were successfully cloned and expressed. In contrast to Aktl（T308E）, Akt1 （T308A） could inhibit Notchl expres- sion and proliferation of drug-resistance ceils. Conclusion： PI3K/Akt and Notch1 signaling pathways plays a key role in gastric cancer drug-resistance development and progression.

关 键 词：AKT1 NOTCH1 点突变 耐药 胃癌 

分 类 号：Q78[生物学—分子生物学] Q23