摘要
目的:构建带myc标签的Six1基因的真核表达载体,获得myc-Six1融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的乳腺文库为模板,采用PCR技术扩增Six1编码序列,将其插入pXJ-40-myc载体,Western印迹检测其在人293T细胞中的表达;将重组质粒与空载体分别转染乳腺癌细胞ZR75-1,通过qRT-PCR检测细胞中VEGF-C在mRNA水平的变化。结果:双酶切和测序结果表明myc-Six1真核表达质粒构建成功;Western印迹证明转染293T细胞后成功表达;qRT-PCR结果表明myc-Six1可升高乳腺癌细胞ZR75-1的VEGF-C转录水平。结论:构建了带myc标签的Six1表达载体,为进一步研究Six1在乳腺癌转移中的作用奠定了基础。
Objective： To construct the eukaryotic expression vector of human Sixl labeled with myc tag and de tect its activity. Methods： Human Sixl gene was obtained from cDNA library by PCR and cloned into pXJ-40- myc vector. Human 293T cells were transfected with the recombinant plasmid of myc-Sixl and the expression was detected by Western blot. VEGF-C expression at the mRNA level regulated by myc-Sixl in breast cancer ZR75- 1 cells was identified by qRT-PCR. Results： Sixl eukaryotic expression vector labeled with myc tag was success fully constructed by double digestion identification. The inserted fragment was confirmed to be correct by sequenc ing. The qRT-PCR showed that human Sixl gene could up-regulate the VEGF-C mRNA expression in breast can cer ZR75-1 ceils. Conclusion： The eukaryotic expression vector myc-Sixl was successfully constructed, and it could up-regulate the mRNA level of VEGF-C, which laid the foundation for the further study of the role of Sixl in breast cancer progression and metastasis.
出处
《生物技术通讯》
CAS
2014年第1期17-20,共4页
Letters in Biotechnology
基金
国家自然科学基金(31100604)
