长白猪CIDEB基因真核表达载体构建及生物信息学分析

本研究旨在从长白猪肝脏组织中克隆与脂质存储相关的CIDEB基因编码区全长序列,并进行生物信息学分析,为研究其结构和功能奠定基础。根据NCBI中的猪CIDEB基因mRNA序列(登录号:NM001112688.1)设计特异性引物,采集长白猪新鲜的肝脏组织,提取RNA并反转录合成cDNA,以cDNA为模板进行PCR扩增,将PCR产物与pcDNA3.1+载体连接,转入大肠杆菌DH5α感受态细胞,筛选阳性克隆后进行生物信息学分析。结果显示:长白猪CIDEB基因编码区序列全长为660 bp,编码了219个氨基酸,与人同源性最高,为92.2%。对长白猪CIDEB蛋白进行生物信息学分析表明,CIDEB蛋白属于亲水性蛋白,不存在跨膜结构,属于CIDE-N家族成员之一,不同物种的CIDEB蛋白具有很高的保守性,其二级结构α-螺旋、延伸链、β-转角和无规则卷曲分别占44.29%、18.26%、8.22%、29.22%。本实验成功构建了CIDEB基因的全长真核表达载体,并对其核苷酸、氨基酸进行序列分析,为进一步研究和揭示长白猪CIDEB蛋白的结构与功能提供了基础材料。

Construction of Eukaryotic Expression Vector and Bioinformatics Analysis of CIDEB Gene in Landrace Pigs

The aim of this study was to clone the full-length coding sequence of related gene in lipid storage from the liver tissue of Landrace pigs and analyze its biological characteristics to lay a foundation for studying its structure and function. Specific primers were designed based on the predicted mRNA sequence of the Landrace CIDEB gene in GenBank(accession number: NM_001112688.1),and the fresh liver tissue samples of Landrace pigs were collected, and RNA was extracted and then was reverse transcribed to synthesize cDNA,and CIDEB gene was amplified by PCR using cDNA as template. The PCR products then were inserted into the pcDNA3.1+ vector,then was transferred to Escherichia coli DH5 competent cells and the requisite clones were filtered, Then analyze its biological characteristics. The results showed that the coding region of CIDEB gene of Landrace was 660 bp in length and encoded 219 amino acids. The sequence of CIDEB gene in Landrace had the highest homology with human beings(92.2%).Bioinformatics analysis of CIDEB gene coding protein in Landrace liver showed that CIDEB protein belonged to hydrophilic liposoluble protein without transmembrane structure and belonged to CIDE-N family. CIDEB protein of different species was highly conservative. Its secondary structure, alpha-helix, elongation chain, beta-rotation and irregular curl accounted for 44.29%, 18.26%, 8.22% and 29.22%.The full-length eukaryotic expression vector of CIDEB was successfully constructed and its nucleotides and amino acids were analyzed, which provided basic materials for further study and revealing the structure and function of CIDEB protein in Landrace.