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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。
OBJECTIVE: To clone a novel and alternative splice variant of type I imidazole receptor (I1R, Nischarin) and to investigate its expression and subcellular localization to explore the difference between its expression and I1R, so as to explain the diversity of pharmacological effects of I1R basis. Methods Primers were designed based on the previous studies of our group. The new alternative splicing was cloned from rat liver tissue by RT-PCR and cloned into pcDNA3.1, pEGFP-N1 and PCMV-myc eukaryotic expression vector And their distribution in 293T cells was explored by cell transfection. The relative molecular mass characteristics of the expressed protein in eukaryotic cells were determined by Western blotting. Results A novel I1R alternative splicing (named I1R-ISO-472) was successfully cloned and encoded a 472 amino acid sequence, which was identical to that of the new gene of GenBank with the sequence of AK036043.1. Bioinformatics analysis showed that this gene is located on chromosome 14B and contains two important functional domains of PX_domainsuperfamily and LRR-RI superfamily. The relative molecular mass of I1R-ISO-472 protein expressed in 293T cells was about 52 × 103, which was consistent with the prediction. Cell immunofluorescence results showed that it was uniformly dispersed in 293T cells. Conclusions A novel I1R alternative splicing clone was successfully cloned. The expression characteristics and subcellular distribution of I1R suggest that its pharmacological action may be different from the known I1R.