目的：构建 UCA1 a（CUDR）基因真核表达载体 pcDNA-UCA1 a（CUDR），观察其在膀胱癌 UM-UC-2细胞中的表达，为研究 UCA1 a（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 UCA1 a（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-UCA1 a（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2200 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-UCA1 a（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-UCA1 a （CUDR）的UM-UC-2细胞中 UCA1 a（CUDR）基因表达量升高。结论：成功构建pcDNA-UCA1 a（CUDR）真核表达载体，且 UCA1 a（CUDR）基因在转染表达载体的 UM-UC-2细胞中高表达。%Objective 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-2 1 1 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-UCA1 a (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.
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