Marc145细胞源EDC3基因的克隆及其生物信息学分析

为获得Marc145细胞源EDC3基因序列，分析其序列特征及编码蛋白的结构和功能，本试验采用RT-PCR方法从Marc145细胞中扩增EDC3基因，并进行克隆和序列测定；应用DNAStar软件分析该基因的核苷酸和氨基酸序列，与参考序列经BLAST比对后分析同源性，并构建系统进化树；利用生物信息学方法对其编码区蛋白进行二级结构、三级结构、B细胞表位、跨膜结构域和信号肽预测。结果显示，Marc145细胞源EDC3基因长度为1 527 bp，共编码507个氨基酸；EDC3基因编码区核苷酸序列与绿猴、猕猴、狒狒、人、倭黑猩猩、马、野猪、虎鲸、绵羊、非洲象和大熊猫的同源性在91.5%~99.2%之间，与非哺乳动物原鸡同源性最低，仅为81.2%；EDC3氨基酸序列与上述物种的同源性在95.3%~99.6%之间，与原鸡的同源性仅为88.8%。系统进化树结果显示，Marc145细胞源EDC3基因与绿猴的亲缘关系最近，其次是灵长类。蛋白结构预测结果表明，EDC3蛋白主要由α-螺旋和无规则卷曲组成，分别为23.38%和47.35%，二级结构与三级结构预测结果相符。该蛋白存在多个B细胞优势抗原表位，无跨膜结构域及信号肽区域。本试验结果可为Marc145细胞源EDC3基因功能的进一步研究提供参考。

Cloning and Bioinformatics Analysis of EDC3 Gene from Marc145 Cells

In order to obtain the sequence of EDC3 gene from Marc145 cell,and analyze the sequence characteristics of EDC3 gene and the structure and function of EDC3 encoding protein,EDC3 gene from Marc145 cells was amplified and cloned by RT-PCR method.The nucleotide and amino acid sequences of EDC3 gene were analyzed by DNAStar software,the homology was analyzed by BLAST,and the phylogenetic tree was constructed.The secondary and tertiary structures,B cell epitopes,transmembrane domain and signal peptide of EDC3 protein were predicted by bioinformatics softwares.The results showed that the length of EDC3 gene was 1 527 bp,which encoded 507 amino acids.The CDS sequence and amino acid sequence of EDC3 gene from Marc145 cell shared 91.5% to 99.2% and 95.3% to 99.6% identity with Chlorocebus sabaeus,Sus scrofa,Pan paniscus,Papio anubis,Macaca mulatta,Homo sapiens,Orcinus orca,Ovis aries,Ailuropoda melanoleuca,Equus caballus and Loxodonta africana,respectively,the shared lowest homology was 81.2% and 88.8% with Gallus gallus.The results of phylogenetic tree showed that EDC3 gene from Marc145 cell was closely relative with Chlorocebus sabaeus,followed by primates.The alpha helix and radom coil were dominant in secondary and tertiary structures,which was 23.38% and 47.35%,respectively.The results predicted that the protein had multiple B cell dominant antigen epitopes,and there was no transmembrane domain and signal peptide region.The results might provide references for further research on the function of EDC3 gene from Marc145 cell.