关键词: 副猪嗜血杆菌; sapA基因; 克隆; 生物信息学分析

Abstract: 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 DH5α competent 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 contained α-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.

Key words: Haemophilus parasuis; sapA gene; cloning; bioinformatics analysis