| Haemophilus parasuis is a common epiphyte of the upper respiratory tract of pigs, which is Gram-negative. It is an opportunistic pathogen, which can cause Glasser’s disease characterized by fibrinous polyserositis, polyarthritis and meningitis. With the developing and expanding of the size of the pig farm, it caused significant economic losses. However, virulence factors of H. parasuis have not been unequivocally defined yet. Lichtensteiger found that90%of H. parasuis could be detected in the neuraminidase activity in1997. The researcher also considered that neuraminidase is an important virulence factor in H. parasuis. With the gensome of H. parasuis SH0165completed, the neuraminidase was identified as nanH.In mang pathogens, neuraminidase is an important virulence factor, related to nutrition, adhesion and the invasion, toxins, and resistance to host innate immunity. However there are less research of neuraminidase. The experiment firstly analysed the neuraminidase sequence. of15reference strains of H. parasuis, and obtained the soluble expression of the active neuraminidase by prokaryotic expression.1. Sequencing and the analysis of the different H. parasuis standard strains of neuraminidaseFor the first time, nanH gene was amplified with15reference strains of H. parasuis genomic DNA with the primers with nanHF/nanHR. All the products of nanH were2007bp, which were668amine acids. The identifinity of nucleotide was99.54%. The identifity of amine acid was99.29%. Although the amino acid sequence of the neuraminidase is highly conserved in the reference strains, the activity of neuraminidase is very different in different reference strains with the neuraminidase assay kit. We found that The activity of neuraminidase of high virulence of serotype5is greater than other low or middle virulent strains except serotype9. It was proved that the virulence of H. parasuis was highly relational to neuraminidase.The nanH gene of H. parasuis is60%identical to Pasteurella muLtocida,is58%identical to Mannheimia haemolytica, is38%identical to Clostridium perfringens, is34%identical to Clostridium sordellii, is31%identical to Salmonella typhimurium. Meanwhile, with the phylogenetic analysis of15reference strains of H. parasuis,we found high-virulence strains of neuraminidase are closed to each other.2.The structure characteristics of neuraminidase of H. parasuisThe research firstly analysised of the structure of neuraminidase of Haemophilus parasuis. The neuraminidase has one RIF(Arg-Ile/Leu-Pro) motif and2Asp-box(Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid). The RIF motif is observed upstream of the first Asp-box in the sialidase domain.The active area includes Glu148, Tyr267, Asp182and an arginine triad constituted by Arg5、Arg163、Arg233. The arginine triad interacts with the carboxyl group of the Neu5Ac substrate. One of the conserved arginine residues is stabilized by the conserved Glu148. The conserved Tyr267acts as a catalytic nucleophile, and the glutamate residue facilitates the nucleophilic attack of the tyrosine by acting as a general base.3.The soluble expression of NanHThe nanH gene was amplified with H. parasuis SH0165genomic DNA and the primers of nanHF/nanHR, which was2007bp.The nanH gene was linked to pET-32a prokaryotic expression vector. Then the recombinant plasmid was transformed into BL21(DE3) pLysS competent cells. The positive clone was picked to culture in18℃。The precipitation was collected and broken by sonication. For the first time, the neuraminidase was soluble expressed and the activity can be assayed. It helps to study the function of neuraminidase. |
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