人YPEL5基因真核表达载体的构建及在食管癌细胞中的表达

Construction of human YPEL5 gene eukaryotic expression vector and its expression in esophageal carcinoma cells

目的构建人YPEL5基因真核表达重组质粒并在食管癌细胞EC9706中表达。方法以人正常组织的cDNA为模板,扩增得到长366bp的YPEL5基因编码序列,将此序列插入到真核表达载体pCDHCD513B的多克隆位点区域中,得到真核表达载体pCDH-CD513B-Flag-YPEL5,经菌落聚合酶链反应(PCR)鉴定后送公司测序。将构建成功的重组质粒转染到人食管癌细胞系EC9706中,利用实时荧光定量PCR和蛋白质免疫印迹(Western Blot)检测YPEL5基因的表达情况。结果成功扩增出YPEL5基因片段,大小为366bp,经双酶切、连接、转化和筛选得到pCDH-CD513B-Flag-YPEL5重组质粒,通过基因测序鉴定显示重组质粒中插入的基因序列与GenBank中的序列一致,转染EC9706细胞2d后,荧光定量PCR和Western Blot显示YPEL5基因的表达明显上调。结论成功构建了pCDH-CD513B-Flag-YPEL5真核表达载体并在食管癌细胞株EC9706中得到表达,从而为进一步研究其在食管癌进展中的功能奠定了基础。

Objective To construct human yippee-like 5（YPEL5） gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells. Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length. Then this sequence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for obtaining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5. After the bacterial colony PCR identification？it was sent to the corporation for testing the sequence. The successfully constructed recombinant plasmid was transfected into human esophageal carcinoma EC9706 cells. The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot. Results The YPEL5 gene segment with 366 bp in length was successfully amplified. pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion, connection, conversion and screening. The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank. After 2 d of transfecting into EC9706 cells,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-regulated. Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 , thus which lays a foundation for studying its function in the progression of esophageal cancer.