人UROC28基因真核表达载体的构建

Construction and expression of human UROC28 eukaryotic expression vector

目的:构建人UROC28基因的真核表达载体,观察其在HEK293细胞中的表达。方法:以PUC-19-UROC28质粒为模板,扩增UROC28基因编码区的全部序列,克隆入真核细胞表达载体pcDNA3中,酶切鉴定目的基因。重组质粒用脂质体转染HEK293细胞,Western blot观察基因表达情况。结果:PCR扩增的特异性片段长度为430bp,以此构建的重组质粒pcDNA3-UROC28,经EcoRⅠ和XbaⅠ双酶切后显示5.4kb和430bp左右的两条片段,测序结果与GenBank中的人UROC28cds序列一致。证明UROC28因已成功克隆到了真核细胞表达载体pcD-NA3中。Western blot证实pcDNA3-UROC28转染HEK293细胞24 h后有UROC28的表达。结论:成功构建了pcD-NA3-UROC28重组真核表达载体,并在HEK293细胞中表达。

Objective： To construct eukaryotic expression vector of human UROC28 gene, and to detect its expression in HEK293 cell lines. Methods： The whole coding sequence of UROC28 gene was amplified from PUC-19-UROC28. The fragment was inserted into eukaryotic expression vector pcDNA3 plasmid. The recombinant plasmid was verified by digestion and DNA sequencing. The expression of UROC28 gene in HEK293 cells was assayed by western blot. Results： The length of specific fragment amplified by PCR was 430 bp, and the recombinant plasmid pcDNA3-UROC28 showed two bands of 5.4 kb and 430 bp by digestion using respective restriction enzymes EcoR I and Xba Ⅰ . The sequence of UROC28 gene was approved or confirmed by blasting to C, enBank. It was suggested that UROC28 gene had been cloned into pcDNA3 vector correctly. Western blot showed that UROC28 gene was expressed, which was detected 24 h after pcDNA3-UROC28 plasmid was transfected into HEK293 cells. Conclusion： The recombinant eukaryotic expression vector pcDNA3-UROC28 was successfully constructed and expressed in HEK293 cell lines.