摘要
目的构建人脆性位点抑癌基因WWOX真核表达载体,并观察其在人胆管癌细胞株QBC939中的表达。方法通过全基因合成的WWOX cDNA为模版,用PCR技术扩增,将扩增片段连接到PUC57质粒,构建克隆载体,鉴定出阳性克隆后用DNA测序法鉴定重组质粒。将克隆载体与pm-Cherry-N1同时用Xho I/BamH I双酶切,构建真核表达载体pmCherry-N1-WWOX,以重组质粒转染QBC939细胞,通过Western blot检测转染细胞中WWOX的表达。结果成功扩增WWOX基因片段,重组质粒的酶切鉴定及测序结果均证明WWOX基因成功克隆到真核表达载体中,Western blot结果证实转染重组质粒后的QBC939细胞WWOX蛋白表达增强。结论成功构建出含人WWOX基因的真核表达质粒,并将重组质粒pmCherry-N1-WWOX转染到QBC939细胞,为研究WWOX基因在肿瘤发生、发展中的作用提供了有效的分子工具。
Objective To construct the eukaryotic expression vector of WW domain containing oxidoreductase（WWOX）gene and observe its expression in human cholangiocarcinoma cell line QBC939.Methods WWOX cDNA was whole-genome synthesized as a template for PCR.The fragment above was amplified with Xho I/BamH I double digestion and was cloned into the eukaryotic expression vector pmCherry-N1.The recombinant vector was identified by DNA sequencing.The expression of WWOX in recombinant plasmid transfected QBC939 cells was detected by Western blot.Results The target gene sequence of recombinant vector pmCherry-N1-WWOX constructed in this experiment was in accordance with the sequence of human WWOX cDNA reported.Western blot confirmed that WWOX protein expression could be increased after transfection.Conclusion The eukaryotic expression vector pmCherry-N1-WWOX has been constructed successfully.The recombinant plasmid transfected into QBC939 cells can enhance expression of WWOX protein which can provide an effective tool for the research of WWOX gene in carcinogenesis.
出处
《安徽医科大学学报》
CAS
北大核心
2011年第4期328-332,共5页
Acta Universitatis Medicinalis Anhui
关键词
基因
肿瘤抑癌
基因表达
胆管肿瘤
genes
tumor suppressor
gene expression
bile duct neoplasms
