大鼠代谢型谷氨酸受体1亚型基因片段的克隆及其在小脑的表达

Cloning of gene fragment of metabotropic glutamate receptor 1 and its expression in rat cerebellum

目的 克隆大鼠代谢型谷氨酸受体 1亚型 (mGluR1 )基因特异片段 ,制备cRNA探针。方法 从Wistar大鼠小脑中提取总RNA ,以RT PCR方法得到预期的 599bp条带 ,将这一片段克隆到pGEM Teasy载体上 ,经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板 ,通过体外转录的方法合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验 ,以检测探针的可靠性。结果 测序证实用RT PCR的方法获得了mGluR1基因特异片段 ,成功地构建了pGEM TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为 1 0ng/ μl及 30ng/ μl。原位杂交实验的结果显示 ,用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆 ,用正义探针杂交无阳性信号。结论 本实验克隆了mGluR1基因特异片段 ,并制备了cRNA探针 ,用大鼠小脑进行的原位杂交实验显示 ,此探针灵敏度高 。

Objective To clone the gene fragment of metabotropic glutamate receptor 1 (mGluR1) and then use it to prepare the cRNA probes of mGluR1. Methods Total RNA was extracted from cerebellum of Wistar rat and a 599bp positive DNA band was obtained by RT PCR method. The fragment of cDNA was then cloned into pGEM T easy vector, the insert was sequenced after restriction enzyme analysis of this newly constructed plasmid pGEM T mGluR1. Then the sense and antisense templates digested from pGEM T mGluR1 plasmid were purified by glassmilk, and used to prepare digoxingen labeled cRNA probes by transcription in vitro. The expression of mGluR1 in rat cerebellum was examined with the probes by in situ hybridization.Results The DNA sequencing confimed the fragment obtained from RT PCR to be correct to code rat mGluR1 gene and pGEM T mGluR1 plasmid was successfully constructed. The concentrations of sense and antisense probes were 10ng/μl and 30ng/μl respectively detected by dot blot hybridization. High hybridization signal of mGluR1 was observed in Purkinjie cell of rat cerebellum with antisense probe but no signal was detected with sense probe. Conclusions We have cloned a gene fragment of mGluR1 and prepared the cRNA probes with digoxingen labeled. Results of in situ hybridization with mGluR1 in rat cerebellum indicate that the probe is sensitive, specific and reliable, which provide a useful tool for study the expression changes of mGluR1 and its relationship with nerve cell damage.