猪胰岛素诱导1基因的cDNA克隆和差异表达及蛋白质序列分析

Cloning and differential expression of insulin induced gene 1 in pig and the analysis on its protein sequence

克隆猪胰岛素诱导1(INSIG1)基因,并对其蛋白质序列进行分析。根据人INSIG1基因的保守序列设计1对引物,以猪总RNA为模板,经RT–PCR获得INSIG1基因序列,通过相对荧光定量PCR分析该基因在不同组织中的表达量;将INSIG1基因序列连接到pMD19–T Simple载体上,用PCR检测阳性克隆后测序,并对克隆得到的基因进行生物信息学分析。结果表明,INSIG1基因在肺中的表达量最高,其次是在肝脏,再次是在脾脏,在心脏中的表达量最低;通过克隆,获得了一段999 bp的序列,其中831 bp的开放阅读框编码276个氨基酸;该蛋白不含信号肽序列,与其他动物INSIG1基因氨基酸序列的一致性在71%以上。

The study aims to clone the pig insulin induced gene 1(INSIG1) and conducts its protein sequences analysis. According to conserved sequence gene INSIG1 in human, a pair of primers was designed. By adopting pig total RNA as template, the INSIG1 gene sequences was cloned using RT–PCR, then the INSIG1 gene was connected to p MD 19–T Simple vector. The positive clone was sequenced after the identification of clone by PCR, and the sequence characterization was performed from biology information analysis software. The results from relative real-time PCR analysis indicated that INSIG1 transcript abundance was the highest in lung, next in liver, spleen, and heart in turn. The experiment obtained a 999 bp sequence, of which included 831 bp open reading frame encoded with 276 amino acids. From the results of bioinformatics analysis showed that the protein does not contain signal peptide sequence, and from the results of amino acid sequence alignment showed that pig INSIG1 had high similarity score(more than 71%) with other animals in Gen Bank.