中文摘要：

在筛选胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae,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,H-NS）。为了进一步研究H-NS对APP生物被膜形成的影响,实验以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,并克隆到原核表达载体pET-28c中获得重组质粒pET28c-hns,转化大肠杆菌（Escherichia coli）BL21（DE3）,经IPTG诱导表达和组氨酸亲和层析柱纯化获得大小约19kD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0.1~0.3μmol/LrH-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0.4μmol/L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。

英文摘要：

The previous STM （signature-tagged mutagenesis） studies found that the disruption of the/ms gene encoding a histone-like nucleoid structuring protein （H-NS） could enhance biofilm formation of Actinobacillus pleuropneumoniae （APP）. To further investigate the effect of H-NS on APP biofilm formation, the 408 bp complete coding sequence of fins 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 pET28c-hns was transformed into Escherichia coli BL21 （DE3）. And a 19 kD 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 biofilms, but strain 4074 did not. Supplemented with 0.1--0.3 μmol/L 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 0.4 μmol/L ofrH-NS was supplemented in both strains. Results indicated that H-NS negatively regulates biofilm formation ofA. pleuropnelunoniae in a dose-dependent manner.