地高辛标记的大鼠NGF RNA探针的制备和应用

制备用地高辛标记的神经生长因子(NGF)的RNA探针并用其研究NGF在海马组织中的表达。设计NGF引物,构建NGF/pGEMT重组质粒,分别用ApaⅠ和SacⅠ进行酶切得到线性化DNA片段,以Sp6和T7聚合酶转录合成酶合成地高辛标记的(dig)正、反义RNA探针。运用点膜杂交的方法检测探针的敏感度;运用该探针的原位杂交法分析NGF在海马中的表达。本研究成功地构建了NGF/pGEMT质粒,获得了正、反义digNGFRNA探针,并应用该探针在海马中观察到NGFmRNA的表达。本研究的结果为深入探讨NGF在海马发育和损伤过程中的表达奠定了基础。

THE PREPARATION OF RNA PROBES FOR NERVE GROWTH FACTOR LABELED WITH DIGOXINGENIN

In order to study the expression of nerve growth factor (NGF), the sense and anti-sense RNA probes for in situ hybridization of NGF were prepared in this study. The NGF fragment which was obtained by RT-PCR was subcloned into pGEM-T Easy, then plasmids were linearized with the restriction enzymes of Apa I and Sal I. Using Sp6 and T7 RNA polymerase, respectively, the digoxigenin (dig) labeled sense and antisense probes were transcriped in vitro, and specificity of sense and antisence dig-NGF RNA probes were confirmed by dot blot. By Using the probe we found that NGF is expressed in hippocampus. In summany, we established an approach to further study the expression of NGF in dereopment and injury of hippocampus.