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目的利用差异表达基因克隆方法(抑制消减杂交)获得大量的动脉粥样硬化相关候选基因和表达序列标签后,探讨如何进行后续基因表达及功能的研究。方法利用Internet网络上的数据库及生物学分析软件对胆固醇损伤内皮细胞后获得的差异表达基因进行核酸序列和蛋白质序列分析,探索差异表达基因克隆后的研究方法和思路。结果通过电子延伸得到一个684bp的全长cDNA序列;通过核酸序列分析,该序列定位在线粒体基因组的7587位～8270位,含有一个完整的开放阅读框,编码与氧化磷酸化相关的9个亚基。对其中一个亚基细胞色素氧化酶Ⅱ(COX2)分析得知,细胞色素氧化酶Ⅱ基因编码一段25.6kDa的弱酸性的信号锚蛋白,细胞色素氧化酶Ⅱ蛋白的三维结构是一个典型的椅式结构,它含有一段疏水区域,一个跨膜结构域和一个胞质结构域。运用蛋白的进化分析,得知细胞色素氧化酶Ⅱ蛋白胞质结构域的氨基酸序列在进化过程中高度保守。结论生物信息学技术是一种高效的获取疾病相关基因信息的方法,利用生物信息学方法对细胞色素氧化酶Ⅱ基因进行分析,获得了基因及其编码蛋白的相关信息,该蛋白参与电子传递,可能与细胞的氧化应激有关。
OBJECTIVE: To obtain a large number of candidate genes related to atherosclerosis and express sequence tags by differential cloning (suppression subtractive hybridization) and to explore how to study the subsequent gene expression and function. Methods The database and biological analysis software were used to analyze the nucleic acid sequence and protein sequence of differentially expressed genes obtained after cholesterol injury of endothelial cells, and to explore the research methods and ideas of differentially expressed genes. As a result, a full-length cDNA sequence of 684bp was obtained by electron extension. The sequence was located at positions 7587-8270 of the mitochondrial genome by nucleic acid sequence analysis and contained a complete open reading frame (ORF) encoding 9 subunits associated with oxidative phosphorylation . One of the subunit cytochrome oxidase Ⅱ (COX2) analysis revealed that cytochrome oxidase Ⅱ gene encodes a 25.6kDa weak acid signal ankyrin, cytochrome oxidase Ⅱ three-dimensional structure of the protein is a typical chair type Structure, which contains a hydrophobic region, a transmembrane domain and a cytoplasmic domain. Using the evolutionary analysis of the protein, we found that the amino acid sequence of the cytoplasmic domain of cytochrome oxidase II is highly conserved during evolution. Conclusion Bioinformatics is an efficient method for obtaining disease-related gene information. The bioinformatics method was used to analyze the gene of cytochrome oxidase Ⅱ, and the related information of the gene and its encoded protein was obtained. The protein involved in electron transport, May be related to cellular oxidative stress.