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构建能在哺乳动物细胞中表达尿激酶受体（ｕＰＡＲ）反义ＲＮＡ的表达质粒ｐＵＲＡＳ。方法采用ＲＴ－ＰＣＲ方法扩增并克隆尿激酶受体（ｕＰＡＲ）ｃＤＮＡ５’端－４６～４５４ｂｐ一段长５００ｂｐ的顺序，利用ＤＮＡ重组技术将该片段反向插入表达载体ｐｃＤＮＡ３的ＣＭＶ启动子下游。结果限制性内切酶分析和ＤＮＡ顺序分析证实ＲＴ－ＰＣＲ获得的ｕＰＡＲｃＤＮＡ片段与文献报道相吻合；重组质粒ｐＵＲＡＳ转染肺癌细胞株９５Ｄ，ＮｏｒｔｈｅｒｎＢｌｏｔ证实转染细胞克隆有ｕＰＡＲ反义ＲＮＡ的表达。结论重组质粒ｐＵＲＡＳ能在肺癌细胞株９５Ｄ中表达尿激酶受体反义ＲＮＡ，尿激酶受体（ｕＰＡＲ）反义ＲＮＡ表达质粒的构建获得成功。
Construction of an expression plasmid pURAS capable of expressing urokinase receptor (uPAR) antisense RNA in mammalian cells. Methods A 500 bp sequence of -46 ~ 454 bp was amplified and cloned by RT-PCR from the 5 ’end of uPAR. The fragment was reverse inserted into the downstream of CMV promoter of pcDNA3 by DNA recombination technique. Results The results of restriction endonuclease analysis and DNA sequence analysis confirmed that the uPAR cDNA fragment obtained by RT-PCR was consistent with that reported in the literature. The recombinant plasmid pURAS was transfected into lung cancer cell line 95D and Northern Blot was used to confirm the expression of uPAR antisense RNA in transfected cells. Conclusion The recombinant plasmid pURAS can express urokinase receptor antisense RNA in lung cancer cell line 95D, and the construction of uPAR antisense RNA expression plasmid is successful.