溶藻弧菌二氢硫辛酰胺脱氢酶基因克隆及其生物信息学分析

Molecular Cloning and Bioinformatics Analysis of Dihydrolipoamide Dehydrogenase Gene from Vibrio alginolyticus

根据溶藻弧菌(Vibrio alginolyticus)二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase,DLD)的基因序列设计1对特异性引物。PCR扩增结果显示,DLD(GenBank登录号AGK62253)全长1 428 bp,共编码475个氨基酸残基。根据推导的氨基酸序列预测其分子质量约为50.998 ku,等电点为5.51。细胞定位、SignalP 4.0、TMHMM Server 2.0和SoftBerry-Psite预测结果显示,DLD位于细胞质中,在第29至30个氨基酸之间存在信号肽,不存在跨膜区,氨基酸序列含有1个Asn-糖基化位点、5个蛋白激酶C磷酸化位点、6个酪蛋白激酶II磷酸化位点等多个活性位点。系统进化树结果显示,溶藻弧菌DLD与副溶血弧菌(V.parahaemolyticus)聚为一簇。用Kyte-Doolittle亲水性参数、Karplus-Schulz柔韧性参数、Emini表面可及性参数和Jameson-Wolf抗原性参数分别预测,DLD可能的B细胞抗原优势表位分别是第5~10、106~110、120~125、158~163和175~180区段。利用SWISS-MODEL软件,模拟DLD亚基三维结构模型,结果显示其与大肠杆菌(Escherichia coli)DLD蛋白有相似构型。Kegg分析发现,其涉及糖酵解和糖异生等9条信号通路。从生物信息学角度验证了DLD作为弧菌共同抗原的可行性。

Primers for PCR cloning were designed according to the whole genome sequence of Vibrio alginolyticus published in GenBank. The dihydrolipoamide dehydrogenase (DLD) gene of V. alginolyticus strain HY9901 was amplified by PCR and cloned into pMD18-T vector to investigate the possibility of DLD as a candidate antigen for vaccine production. Sequence analysis revealed DLD gene (GenBank Number: AGK62253) is 1 428 bp and encodes a putative protein of 475 amino acids. The predicted molecular mass of DLD was 50.998 ku with an estimated pI of 5.51. Using SignalP 4.0 and TMHMM Server 2.0 software, it was predicted that the DLD protein did not contain a signal peptide or a transmembranous region. This protein had one asn-glycosylation site, five protein kinase C phosphorylation site, six casein kinase II phosphorylation site, and so on. To further analyze the evolutionary relationship among DLD, a molecular phylogenetic tree was constructed by using Mega 5.0 software. In this tree, the DLD protein showed high genetic relationship with Vibrio parahaemolyticus. Using Kyte Doolittle-Hydrophilic parameters, Karplus-Schulz flexibility, Emini surface accessibility and antigenic Jameson-Wolf parameters methods, the B-cell preponderant epitopes of DLD might be localized in the regions of 5-10, 106-110, 120-125, 158-163 and 175-180. The three-dimensional structure of DLD was determined by using SWISS-MODEL work-space and it had a similar structure to DLD protein of Escherichia coli. Kegg analysis found that DLD involved nine signaling pathways, such as sugar glycolysis and dysplasia, and so on. These results can provide a basis for further studies on the shared immunogenecity of DLD and vaccine preparation.