副猪嗜血杆菌sapA基因克隆与生物信息学分析

Cloning and Bioinformatics Analysis of sapA Gene of Haemophilus parasuis

试验旨在克隆副猪嗜血杆菌(Haemophilus parasuis,Hps)sapA基因,并对其进行生物信息学分析,以期为sapA基因缺失疫苗的开发与应用提供理论依据。以71株临床菌株为研究对象,经PCR扩增鉴定其中的阳性菌株;将扩增得到的sapA基因片段与pGEM-T载体连接,转化大肠杆菌DH5α感受态细胞,经PCR和双酶切鉴定为阳性克隆后进行测序;用生物信息学软件进行核苷酸与氨基酸序列比对、系统进化树分析、蛋白二级和三级结构预测、疏水性预测、柔性区域位预测、B细胞抗原表位预测和Jameson-Wolf抗原指数预测。试验结果显示,在71株临床菌株中,有29株成功扩增出sapA基因,29株Hps的sapA基因核苷酸序列与已公布的SH0165菌株相似性为96.5%~98.8%,除H88菌株外,氨基酸序列与SH0165菌株相似性为98.9%~100%。sapA蛋白二级结构主要有α-螺旋、β-转角和无规则卷曲,有13个亲水区、38个柔性区和23个B细胞潜在抗原表位。以上结果表明,Hps的sapA基因是一个保守基因,各菌株间核苷酸序列、氨基酸序列都有很高的相似性,sapA蛋白具有较强的抗原性。

The aim of this study was to clone and analyze bioinformatics of the sapA gene of Haemophilus parasuis(Hps),and provide theoretical basis for the development and application of sapA gene deletion vaccine.71 clinical strains were selected as the research objects,and the positive strains were identified by PCR amplification.The cloned sapA gene fragments were linked with pGEM-T vector and transformed into DH5αcompetent cells of Escherichia coli.The positive clones were identified by PCR and double enzyme digestion and then sequenced.Bioinformatics softwares were used for nucleotide sequence and amino acid sequence alignment,phylogenetic tree analysis,protein secondary structure and tertiary structure prediction,amino acid hydrophobicity prediction,flexible region prediction,B cell epitope prediction and Jameson-Wolf antigen index prediction analysis.The results showed that sapA gene was successfully amplified in 29 out of 71 clinical isolates.The sequence of sapA gene of 29 Hps strains shared 96.5%-98.8%similarity with the published SH0165 strain.The amino acid sequence of sapA gene of 28 Hps strains shared 98.9%-100%similarity with SH0165 strain except for the H88 strain.The secondary structure of sapA protein containedα-helix,β-turn and random coil,with 13 hydrophilic regions and 38 flexible regions,23 potential sites on the surface of B cell antigen.The above results indicated that the sapA gene of Hps was a conserved gene with high homology in nucleotide sequence,amino acid sequence,and the sapA protein had strong antigenicity.