地高辛精标记大鼠代谢型谷氨酸受体第5亚型RNA探针的制备研究

A STUDY ON PREPARATION OF DIGOXIGENIN LABELLED RAT METABOTROPIC GLUTAMATE RECEPTOR 5 RNA PROBE

为制备用地高辛精标记的大鼠代谢型谷氨酸受体第 5亚型 c RNA探针 ,本实验用分子生物学技术重组质粒 p GEMm Glu R5 ,经限制性内切酶酶切分析 ,证实其确有 m Glu R5基因片段插入且方向正确。将此质粒再经限制性内切酶消化得到线性DNA片段 ,用 T7RNA聚合酶体外转录合成带有地高辛精标记的高比活性的单链 RNA探针 ;经斑点杂交实验证实该探针具有较高的敏感性和可靠性 ,可用于代谢型谷氨酸受体第

To prepare a digoxingen labelled cRNA probe for rat metabotropic glutamate receptor 5,the recombinant plasmid pGEM mGluR5 was constructed by using molecular cloning techniques.According to analysis of restriction endonucleases, the plasmid pGEM mGluR5 contained the DNA fragment of rat mGluR5 gene and its insertion direction was correct. The plasmid pGEM mGluR5 was used as a template to synthesize the digoxigenin labelled RNA probe with a transcription procedure in vitro. The probe was identified by dot blot hybridization. The results indicat the probe prepared in the present study is very sensitive and reliable, and it may be useful for the study of mGluR5.