Molecule cloning and analysis of tissue expression profile of porcine Gpr6 gene | 猪Gpr6基因的克隆及表达分析

Chinese. 为了探明G蛋白偶联受体6基因（Gpr6）的结构、功能与表达模式,克隆了含有完整阅读框的猪Gpr6的cDNA序列。利用生物信息学方法对Gpr6基因的结构及功能进行了系统研究,运用Real-time PCR技术对其在组织中的表达规律进行了分析,并利用构建的原核表达载体pET32a-Gpr6对其在BL21菌株中的最佳蛋白表达条件进行了筛选。结果表明：猪Gpr6基因的cDNA序列由1 664个核苷酸组成,编码366个氨基酸残基,等电点为7.63,相对分子质量为38.38×103,与牛、犬、人、黑猩猩、大鼠和小鼠的氨基酸相似性分别为91.2%、89.5%、88.3%、88.3%、86.3%和86.1%。猪Gpr6基因在各组织中均有表达,但仅在下丘脑、垂体和肝脏中表达量较高。经原核表达分析,发现成功获得了相对分子质量约为27 000的Gpr6蛋白,并且确定了该蛋白的最佳表达条件为0.8 mmol.L-1IPTG,37℃诱导表达2 h。

English. In this paper, the complete cDNA sequence of porcine Gpr6 gene was cloned using RACE method and the expression pattern of the gene in various tissues was investigated using real-time PCR. Prokaryotic expression vector pET32a-Gpr6 was constructed and the conditions of its protein expression in BI21 strains were screened. The results indicated that the complete sequence of Gpr6 gene with its length being 1 644 bp nucleotides, encoded a polypeptide of 366 amino acids with the relative molecular weight of 38.38× 10. and an isoelectric point of 7.63. Comparison of the putative amino acids of porcine orthologs with those of other species showed that porcine Gpr6 shared 91.2% ,89.5% ,88.3% ,88.3% ,86.3% and 86. 1% homology with Bos taurus, Canis lupus familiaris ,Homo sapiens, Pan troglodytes, Mus musculus and Rattus norvegicus respectively. Porcine Gpr6 mRNA was found expressed almost in all tissues, highly in hypothalamus and pituitary, and hardly in liver. The result from prokaryotic expression analysis indicated that Gpr6 protein of approximately 27 ×10. was obtained and optimum condition for the expression of the protein was 0.8 mmol. L-1 IPTG,37 ℃ ,2 h.