知识与财富的链接
目的:构建稳定表达p120ctn的A549细胞株,以研究p120ctn蛋白在肺癌发生和转移过程中的作用。方法:通过分子克隆,将pc DNA3.1多克隆位点插入Flag标签的编码序列,得到pc DNA.Flag表达载体。然后PCR扩增p120ctn的编码序列,插入Flag标签下游,构建pc DNA.Flag-p120ctn质粒,筛选阳性克隆并进行酶切及测序鉴定。利用脂质体Lipofectamine 2000将pc DNA.Flag-p120ctn质粒转染到肺癌细胞A549中,通过G418筛选得到稳定转染细胞株,免疫印迹法检测p120ctn的表达。结果:本文构建了融合有Flag标签的p120ctn真核表达载体并转染到A549中,免疫印迹结果表明p120ctn蛋白在A549细胞中高效的表达。结论:本文成功构建了稳定高表达p120ctn的A549细胞模型,为深入研究p120ctn在肺癌的发生和转移过程中的作用奠定了基础。
Objective:To establish a stable A549 cell line over-expressing p120ctn through pcDNA3.1.Methods:First, a DNA fragment encoding Flag-tag was synthesized. A p1 20ctn gene was cloned by PCR and inserted into the multiple cloning sites of pcDNA3. 1 to construct the plasmid pcDNA.Flag-p120ctn. The vector was identified by enzyme digestion and DNA sequencing. Secondly, we transfected pcDNA.Flag-p120ctn into A549 cell by lipofectamine 2000. Then, stable transfected A549 cell line expressing p120ctn was established after screening culture by G41 8 and was identified by Western blot.Results:The plasmid pcDNA. Flag-p120ctn was constructed. Stable transfected A549 cell line was established and p1 20ctn was over-expressed successfully.Conclusion:We successfully generate a stable A549 cell line expressing p1 20ctn which sets a cell model for further research into the genesis and development of human lung adenocarcinoma.
