摘要
构建人源细胞周期检测点蛋白RAD9A基因真核细胞表达载体,转染至肾癌细胞786-0中,使EGFP-RAD9A融合蛋白得到高效表达。通过PCR法从293T细胞c DNA中扩增RAD9A基因编码区,经双酶切后连接入表达载体p EGFP-N1多克隆位点,并对阳性重组子进行鉴定。将p EGFPRAD9A转染至肾癌细胞786-0中,荧光显微镜观察GFP-RAD9A融合蛋白的表达及亚细胞定位,Western blot免疫印记法检测转染p EGFP-RAD9A后的肾癌细胞786-0中的融合蛋白。通过定向克隆的方法获得了含有RAD9A基因编码区的真核表达载体p EGFP-RAD9A。与对照组相比,转染p EGFP-RAD9A的肾癌786-0细胞中高效表达了EGFP-RAD9A融合蛋白,后者定位在786-0的细胞核。构建的RAD9A基因重组真核表达载体能够在肾癌细胞786-0中高效表达,为研究RAD9A在肾癌发生发展中的作用奠定了基础。
Objective： to construct the human cell cycle checkpoint protein RAD9 A gene in eukaryotic expression vector,transfect the plasmid into renal carcinoma cell line 786- 0 and explore the high expression of EGFP- RAD9 A fusion protein. Methods： Amplified RAD9 A gene protein- coding region by PCR from of293 T c DNA. After restrictive enzyme digestion,this fragment was inserted into multiple clone site of p EGFP-N1 vector. Then the positive ecombinants were identified by PCR and DNA sequencing analysis. The plasmid p EGFP- RAD9 A was transfected into renal carcinoma cell line 786- 0 to explore the expression and subcellar localization of GFP- RAD9 A fusion protein by fluorescence microscope. Then the Western blot was further used to detect the expression of GFP- RAD9 A fusion protein in 786- 0 cell. Results： recombinant eukaryotic expression vector p EGFP- RAD9 A was obtained by directed cloning of RAD9 A gene. Compared with the control group,the EGFP- RAD9 A fusion protein expressed efficiently in transfected 786- 0 cell line,and was identified in the nucleus of 786- 0 cell. Conclusion： The eukaryotic expression vector p EGFP-RAD9 A was successfully constructed and highly effective expressed in 786- 0 cells,which can be used for the further research on the function of RAD9 A gene in progression of renal cell carcinoma（ RCC）.
出处
《湖北理工学院学报》
2016年第6期56-60,共5页
Journal of Hubei Polytechnic University
基金
湖北理工学院大学生科技创新项目(项目编号:201510920019)
湖北理工学院人才引进项目(项目编号:16xjz08R)
湖北省教育厅科学技术研究计划青年人才项目(项目编号:000713)
关键词
周期检测点蛋白RAD9A
定向克隆
真核细胞表达载体
肾癌
cell cycle checkpoint protein RAD9A
directed cloning
eukaryotic expression vector
renal cell carcinoma（RCC）
