地高辛标记的大鼠nNOS mRNA探针的制备和应用

The preparation of RNA probes of neuronal nitric oxide synthase labeled by digoxingenin

目的 制备大鼠神经元型一氧化氮合酶 (neuronalnitricoxidesynthase,nNOS)地高辛 (digoxigenin)标记的RNA探针 ,探讨nNOS在脊髓中的表达定位。方法 采用RT PCR方法 ,从大鼠脑组织中扩增nNOS基因mRNA部分片段 ,并经序列测定。以dig nNOSmRNA为探针 ,采用原位杂交观察成年大鼠脊髓组织中nNOSmRNA表达。结果 RT PCR法扩增出一特异产物与预期长度 2 4 0bp相符 ,T载体克隆测序与nNOS基因 10 0 %同源。原位杂交结果显示阳性信号出现在成年大鼠脊髓组织中。结论 采用RT PCR和T载体技术获得了大鼠脑组织nNOS基因克隆 ,dig nNOSmRNA探针原位杂交显示正常SD大鼠腰段脊髓组织中表达nNOSmRNA。

Objective To obtain the clone of neuronal nitric oxide synthase(nNOS)from rat brain and then labeled mRNA probe by digoxigenin.Methods The partial mRNA fragment of nNOS was amplified by RT PCR from rat brain.RT PCR product was ligated into pGEM T vector,and was sequenced.The expression of nNOS mRNA in lumbar spinal cord of adult rat was examined by in situ hybridization with dig nNOS mRNA.Results The product of RT PCR was the 240 bp which matched the length expected.The sequence of nNOS was completely homogeneous with the nNOS sequence reported.Hybridized signals of nNOS mRNA were found in lumbar spinal cord of the normal SD rat.Conclusions nNOS gene was obtained from rat brain using RT PCR and T vector techniques.The study of in situ hybridization using dig nNOS mRNA indicated that the nNOS mRNA expressed in the lumbar spinal cord of the normal SD rat.