一个新的Ⅰ型咪唑啉受体可变剪接体的克隆、鉴定及亚细胞分布

目的克隆Ⅰ型咪唑啉受体（I1R,Nischarin）新的可变剪接体,并对其进行表达特征和亚细胞定位研究,探索其表达与I1R的差异,从而为解释I1R药理学作用多样性奠定基础。方法在本课题组前期研究基础上设计引物,通过RT-PCR从大鼠肝组织中克隆新的可变剪接体,将其克隆到pcDNA3.1、pEGFP-N1和PCMV-myc3个真核表达载体中,并通过细胞转染探索其在293T细胞中的分布,应用Western印迹测定其在真核细胞中表达蛋白的相对分子质量特征。结果成功克隆获得一个新的I1R可变剪接体（命名为I1R-ISO-472）,编码472个氨基酸,与GenBank中序列号AK036043.1的新基因的序列完全一致。生物信息学分析表明,该基因染色体定位于14号染色体14B,含有磷酯酰肌醇结合位点（PX_domainsuperfamily）与亮氨酸富集重复（LRR-RIsuperfamily）两个重要的功能结构域。I1R-ISO-472在293T细胞中表达的蛋白相对分子质量约为52×103,与预测基本一致。细胞免疫荧光结果表明,其在293T细胞内呈均匀弥散分布。结论成功克隆一个新的I1R可变剪接体,其表达特征及亚细胞分布提示其药理学作用可能与已知的I1R不同。

Cloning, identification and cellular localization of a new isoform of I1R

Objective To clone a novel I1R isoform and explore its relative molecular mass and subcellular location in eukaryotic cells so as to learn about its role in the pharmacological function of I1R.Methods On the basis of our previous results,we chose complete CDS AK036043.1 to amplify the I1R-ISO-472 spliced variant by RT-PCR from the mice liver.The product was cloned into pcDNA3.1,PCMV-myc and pEGFP-N1 vectors.After restriction enzyme digestion analysis and sequencing,these recombinant plasmids were transfected into 293T cells and observed with the inverted fluorescence microscope.Then its relative molecular weight mass was analyzed by Western-blot.Results We gained a 1420 bp cDNA fragment （AK036043.1） which was expressed in the eukaryotic cell and translated a protein containing 472 amino acid residues.The sequence was the same as AK036043.1 in GenBank.Using bioinformatics I1R-ISO-472 was mapped to chromosomes 14B,and its protein contained two important domains including the PX_domain superfamily and LRR-RI superfamily.In 293T cells,the subcellular location of protein was detected in cytoplasm.Conclusion A new isoform of I1R is obtained.According to its relative molecular mass and subcellular location,its pharmacological function may be different from that of I1R known.These results help study the new biological function of I1R,providing information for clarifying the role of I1R spliced variant in its pharmacological function.