大鼠脑组织中p75基因克隆及其探针制备

为了克隆大鼠p75基因并制备地高辛标记的p75 cDNA探针(dig-p75 cDNA),本研究采用RT-PCR法,从大鼠脑组织mRNA中扩增p75基因mRNA部分片段,克隆入T载体,并经序列测定。以dig-p75 cDNA为探针,采用原位杂交方法观察成年SD大鼠海马组织中p75 mRNA的表达。结果:RT-PCR法扩增出一种特异产物与预期长度386 bp相符,T载体克隆测序与p75基因100％同源。原位杂交结果显示,阳性信号出现在成年大鼠海马组织中。结论:采用RT-PCR和T载体技术获得了大鼠脑组织p75基因克隆,dig-p75 cDNA探针原位杂交显示正常SD大鼠海马组织中表达p75 mRNA。

GENE CLONING OF p75 FROM RAT BRAIN AND ITS PROBE LABELED

To obtain the clone of p75 from rat brain and then labeled cDNA probe by digoxigenin, the partial mRNA fragment of p75 was amplified by RT-PCR from rat brain. RT-PCR product was ligated into pGEM-T vector, and was sequenced. The expression of p75 mRNA in hippocampus of adult rat was examined by in situ hybridization with dig-p75 cDNA. The product of RT-PCR was the 386 bp which matched the length expected. The sequence of p75 was completely homogeneous with the p75 sequence reported. Hybridized signals of p75 mRNA were found in hippocampus of the SD rat. The results show that p75 gene can be obtained from rat brain using RT-PCR and T vector techniques. The study of in situ hybridization using dig-p75 cDNA indicates that the p75 mRNA is expressed in hippocampus of SD rat.