5株内阿米巴14-3-3蛋白序列比较及生物信息学分析

Comparison of 14-3-3 proteins in 5 Entamoeba strains and their relative bioinformatics analysis

目的比较具有不同致病性以及毒力的5株内阿米巴的14-3-3蛋白序列,并选取溶组织内阿米巴HM1∶IMSS株进行相关生物信息学预测,用以指导进一步实验研究。方法收集各虫株滋养体的基因组DNA,根据Gen-Bank收录的溶组织内阿米巴编码基因序列设计特异引物,以基因组DNA为模板扩增目的基因片段,测序后利用生物信息学网站的各种在线分析工具和Genetyx软件ver 13.0,对所得序列进行比较,构建分子进化树,并对溶组织内阿米巴HM1∶IMSS株的14-3-3蛋白进行相关的生物信息学分析。结果5株内阿米巴属虫株均含有3个14-3-3基因,编码的氨基酸序列同源性较高,个别位点存在差异。取溶组织内阿米巴HM1∶IMSS株14-3-3-1序列与其他物种的同源蛋白比较并构建分子进化树,与种系进化过程非常吻合。根据生物信息学分析结果预测,溶组织内阿米巴HM1∶IMSS株14-3-3-1含720个碱基,编码239个氨基酸;14-3-3-2含717个碱基,编码238个氨基酸;14-3-3-3含723个碱基,编码240个氨基酸。3种异构体都带有2个14-3-3蛋白家族标记,含有多个潜在的磷酸化位点,但不含线粒体、过氧化物酶体等细胞器定位序列以及信号肽。该蛋白在大肠埃希菌中表达的半衰期>10 h。结论内阿米巴属14-3-3基因高度保守。生物信息学分析结果提示14-3-3蛋白是研究物种进化的理想分子。

Objective To compare the sequences of 14-3-3 proteins in the Entamoeba histolytica HM1 ： IMSS strain with those in another four Entamoeba strains and predict their structure and function. Methods Genomic DNA was isolated from different Entamoeba strains. Specific primers were designed according to the E. histolytica 14-3-3 sequences at GenBank, and the 14-3-3 genes were amplified by PCR. By employing tools at online bioinformatics websites and software packages such as Genetyx, the sequences obtained were compared, and a phylogenetics tree was constructed. The bioinformatics prediction of the E. histolytica 14-3-3 proteins has also been performed. Results There are three 14-3-3 isoforms in the five Entamoeba strains used in the study. These isoforms＇amino acid sequences show high homology between strains. The phylogenetic analysis indicated that the evolution of the 14-3-3 gene was relative to the evolution of these species. Bioinformatics prediction shows that the 14-3-3-1 isoform contains 723 bp coding for 239 amino acids, the 14-3-3-2 isoforms contain 717 bp coding for 238 amino acids and the 14-3-3-3 isoforms contain 720 bp coding for 240 amino acids. These isoforms have many phospholylation sites and two 14-3-3 signatures. However, they do not have signal peptide or cell organelle targeting peptide such as mitochondrial targeting peptide and peroxisome targeting peptide. Besides, the estimated half-time of in vivo expression of these isoforms in Escherichia coli was longer than 10 hours. Conclusion The 14-3-3 genes are highly conserved in Entamoeba and the 14-3-3 protein can be used as a phylogenetic marker.