重组H-NS蛋白对胸膜肺炎放线杆菌生物被膜形成的影响

摘要：胸膜肺炎放线杆菌（APP）是重要的猪呼吸道病原菌，给世界养猪业造成严重的经济损失。生物被膜在许多细菌的感染与致病过程中起重要作用。我们在筛选APP转座突变体库时，发现hns基因缺失突变可显著增强APP血清1型生物被膜的形成，该基因编码一种DNA结合蛋白（组蛋白样类核结构蛋白）。为了进一步研究H-NS蛋白对APP生物被膜形成的影响，本文以APP血清1型4074株基因组DNA为模板，PCR扩增了408bp的hns基因编码区，克隆到原核表达载体pET-28c中获得重组质粒pET-hns，转化大肠杆菌BL21(DE3)，经IPTG诱导表达和组氨酸亲和层析柱纯化获得大小约19KDa的重组蛋白rH-NS。将不同浓度的rH-NS添加到hns突变株1-21及其亲本菌株4074的培养基中，用微孔板法测定生物被膜的形成。结果显示，在不添加rH-NS时，4074株不能形成可见的生物被膜，而1-21株形成明显的生物被膜；在添加0.1-0.3μM的rH-NS的情况下，1-21株生物被膜的形成量随着rH-NS浓度的升高而降低，而4074株随着rH-NS浓度的升高而升高；rH-NS添加量超过0.4μM对两个菌株生物被膜形成未见明显影响。结果表明H-NS蛋白负调控APP生物被膜的形成，并呈现一定的剂量效应。

Effect of the Recombinant H-NS Protein on Biofilm Formation in Actionbacillus pleuroneumoniae

Abstract: Actinobacillus pleuropneumoniae (APP) is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Biofilm formation contributes to full virulence in many bacterial pathogens. In our previous STM studies we found that the disruption of the hns gene encoding a histone-like nucleoid structuring protein (H-NS) could enhance biofilm formation of APP. To further investigate the effect of H-NS on APP biofilm formation, the 408 bp complete coding sequence of hns gene was amplified by PCR from the genomic DNA of APP serotype 1 strain 4074 and cloned into the prokaryotic expression vector pET-28c. The resultant recombinant plasmid pET-hns was transformed into Escherichia coli BL21 (DE3). A 19KDa of recombinant protein (rH-NS) was obtained by IPTG induction, and purified using the Ni-NTA agarose columns. Different concentrations of rH-NS were added into the culture medium of the hns transposal mutant strain 1-21 and its parental strain 4074, and biofilms were quantified using the microtiter plate biofilm assay. Without rH-NS in the medium, strain 1-21 formed obvious biofims, but strain 4074 did not. Supplemented with 0.1-0.3μM rH-NS in the culture medium, the biomass of biofilms formed by strain 1-21 continuously decreased, whereas that formed by strain 4074 was increased. No obvious changes could be observed when more than 0.4μM of rH-NS was supplemented in both strains. Our data indicated that H-NS negatively regulates biofilm formation of A. pleuropneumoniae in a dose-dependent manner.