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UCA1a(CUDR) 基因真核表达载体的构建及其在膀胱癌UM-UC-2 细胞中的表达

王宇1，陈葳2，李旭2，张红1，张晓芹1，史迎莉1   

  1. （1. 陕西中医学院医学科研实验中心，陕西 咸阳 712046；2. 西安交通大学医学院第一附属医院转化医学中心，陕西 西安710061）
  • 收稿日期:2013-08-30 出版日期:2014-05-28 发布日期:2014-05-28
  • 通讯作者: 李 旭 E-mail:（Tel:029-85323528，E-mail:lixu1956@gmail.com）
  • 作者简介:王 宇（1982-），男，陕西省宝鸡市人，讲师，医学博士，主要从事肿瘤分子遗传学研究。 
  • 基金资助:

    国家自然科学基金资助课题（81372151）；陕西省教育厅科学研究计划(自然科学专项)项目资助课题 (2013JK0765)

Construction of eukaryotic expression vector of UCA1a(CUDR) gene and its expression in bladder cancer UM-UC-2 cells

WANG Yu1，CHEN Wei2，LI Xu2，ZHANG Hong1，ZHANG Xiao-qin1，SHI Ying-li1   

  1. (1. Medical Experiment Center,Shaanxi University of Chinese Medicine,Xianyang 712046,China;2. Center for Translational Medicine,First Affiliated Hospital,College of Medical Sciences,Xi’an Jiaotong University,Xi’an 710061,China)
  • Received:2013-08-30 Online:2014-05-28 Published:2014-05-28

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摘要：

目的：构建 UCA1a(CUDR) 基因真核表达载体 pcDNA-UCA1a(CUDR)，观察其在膀胱癌 UM-UC-2 细胞中的表达，为研究 UCA1a(CUDR) 基因与膀胱癌的关系提供实验依据。方法：以膀胱癌 BLZ-211 细胞的 5′-RACE-Ready cDNA 为模板，采用 PCR 法克隆 UCA1a(CUDR) 全基因，经EcoRⅠ和 BamHⅠ双酶切后与真核表达载体 pcDNA3.1(＋) 连接，构建 pcDNA-UCA1a(CUDR) 重组质粒。双酶切及测序鉴定后，稳定转染至体外培养的人膀胱癌 UM-UC-2 细胞系，利用 RT-PCR法检测转染 pcDNA-UCA1a(CUDR) 的 UM-UC-2 细胞和转染空质粒 pcDNA3.1(＋) 的 UM-UC-2 细胞中 UCA1a(CUDR) 基因的表达。结果：克隆的目的基因片段约为 2 200 bp，与预期结果相符，表明成功扩增 UCA1a(CUDR) 基因；经双酶切及测序鉴定，成功构建真核表达载体pcDNA-UCA1a(CUDR)。半定量 RT-PCR 法检测，与转染空质粒的细胞比较，转染表达载体 pcDNA-UCA1a(CUDR) 的UM-UC-2 细胞中 UCA1a(CUDR) 基因表达量升高。结论：成功构建pcDNA-UCA1a(CUDR)真核表达载体，且UCA1a(CUDR) 基因在转染表达载体的 UM-UC-2 细胞中高表达。

关键词:  , UCA1a(CUDR)基因, 真核表达载体, 转染, UM-UC-2 细胞系

Abstract:

To construct an eukaryotic expression vector pcDNA-UCA1a(CUDR) and to observe its expression in bladder cancer UM-UC-2 cells,and to provide experimental basis for study on the relationship between UCA1a(CUDR) gene and bladder cancer.Methods Human total length of UCA1a(CUDR) gene was obtained from the 5′-RACE-Ready cDNA of bladder cancer BLZ-211 cells by PCR and was inserted into pcDNA3.1(＋) vector.pcDNA-UCA1a(CUDR) was identified by digestion with EcoRⅠ and BamHⅠ.The bladder cancer UM-UC-2 cells were transfected stably with the constructed eukaryotic expression vector pcDNA-UCA1a(CUDR).The expressions of UCA1a(CUDR) gene in the UM-UC-2 cells transfected with pcDNA-UCA1a(CUDR) and the UM-UC-2 cells transfected with pcDNA3.1(＋) (control vector) were detected by RT-PCR. Results The inserted fragment with 2 200 bp was successfully amplified,which was in accordance with the expected results.The eukaryotic expression vector pcDNA-UCA1a(CUDR) was constructed successfully after identified by double enzyme digestion and sequencing.The RT-PCR results showed that the expression of UCA1a(CUDR) gene in the cells transfected with pcDNA-UCA1a(CUDR) was significantly increased compared with the cells transfected with pcDNA3.1(＋).Conclusion The eukaryotic expression vector pcDNA-UCA1a(CUDR) is successfully constructed.The UCA1a(CUDR) gene highly expresses in the UM-UC-2 cells transfected with the expression vector.

Key words: UCA1a(CUDR) gene, eukaryotic expression vector, transfection, UM-UC-2 cell line

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