诱生性一氧化氮合酶cRNA探针的制备及敏感性检测

THE PREPARATION AND DETECTION OF INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) cRNA PROBE

目的制备地高辛标记的大鼠诱生性一氧化氮合酶（ｉＮＯＳ）ｃＲＮＡ探针。方法首先对携带大鼠ｉＮＯＳｃＤＮＡ的ＰＭＯＳＢｌｕｅ质粒进行转化扩增。然后提取扩增的质粒ＤＮＡ并用ＢａｍＨⅠ内切酶酶切处理使其线性化。以线性化的ｃＤＮＡ为模板，在Ｔ７ＲＮＡ聚合酶的作用下，体外转录带有地高辛的ｉＮＯＳｃＲＮＡ探针。结果该地高辛标记的ｃＲＮＡ探针用点杂交法能检出相应ＤＮＡ的敏感性可达０．１μｇ／Ｌ。结论该ｉＮＯＳｃＲＮＡ探针可用作检测组织细胞内ｉＮＯＳｍＲＮＡ的表达。

Objective To prepare DIG labelled iNOS cRNA probe of rat. Methods First, the plasmid (PMOS Blue) carrying iNOS cDNA of rat was proliferated in E.coli HB101. Then, the DNA of the plasmid was isolated and linearized with Bam H restriction enzyme, Finally, the probe of iNOS cRNA was synthesized in vitro with T7 RNA polymerase in the presence of the template (the linearized cDNA) and DIG-UTP. Results The 0.1g/L (0.1pg/l) homologous cDNA was detected with the DIG labelled iNOS cRNA probe in dot-blot. Conclusion The iNOS cRNA probe thus prepared can be used for detecting the expression of iNOS mRNA in tissue cells.