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副猪嗜血杆菌HAPS_ 2096基因介导的天蚕素B抗性研究

发布时间:2018-03-08 14:18

  本文选题:副猪嗜血杆菌 切入点:天蚕素B 出处:《华中农业大学》2015年硕士论文 论文类型:学位论文


【摘要】:副猪嗜血杆菌(Haemophilus parasuis,HPS)是猪的一种常见病原菌,可引起猪的格拉泽氏病(Gl?sser disease),不同年龄阶段的猪均可感染,临床上血清型众多,不易控制,且常常与其他病毒混合感染。在临床治疗上,抗生素是该病防控的首选,而抗生素的滥用更加速了副猪嗜血杆菌耐药性的产生。天蚕素B(Cecropin B,CB)是从惜古比天蚕中发现的具有广谱抗菌活性的天然昆虫抗菌肽(Antimicrobial Peptide,AMP),是一种潜在的治疗感染性疾病的候选药物,具有极大的研发价值。在前期的研究中,我们发现经CB诱导后,副猪嗜血杆菌可产生耐药性,并且与一些基因有关。基因HAPS_2096编码产物为副猪嗜血杆菌ABC型氨基酸转运泵周质底物结合蛋白(Periplasmic substrate binding protein,PSBP),ABC转运泵家族在细菌耐药机制中十分关键。本研究证实了HAPS_2096在副猪嗜血杆菌对CB的抗性中起到重要作用,并且试图探索该基因介导耐药的相关机制。1.HAPS_2096基因缺失株及缺失互补株的构建根据同源基因交叉互换的原理,构建具有与HAPS_2096基因有相同同源臂的自杀载体p K-kan,自然转化HPS0135,获得具有卡那霉素抗性的HAPS_2096基因缺失株。将含有HAPS_2096启动子及其整个开放阅读框的基因片段克隆到经实验室改造过的内源载体p SHk3上,电转化到基因缺失株中,同时转化相应空载体作为对照株。经极性效应、PCR、RT-PCR及基因序列Blast验证上述各菌株构建成功。测定生长曲线发现各菌株生长无明显差异。2.各菌株对CB敏感性试验根据CB对HPS最小抑菌浓度(MIC)设置一系列CB反应浓度,细菌数量为105CFU/m L。基因缺失株和野生株与CB作用1h后,当CB终浓度为0.25μg/m L时,JS0135的存活率为12.02%,显著高于ΔHAPS2096的存活率5.16%(p0.05);同样,当CB终浓度为0.125μg/m L时,JS0135的存活率为26.62%,显著高于ΔHAPS2096的存活率13.09%(p0.05)选择终浓度0.25μg/m L的CB,作用1h后,在该浓度下,含空载体对照株存活率为1.81%,显著低于互补株的存活率6.82%(p0.05)。在琼脂扩散试验中,将1μg/m L的CB溶液加入琼脂孔中,37℃孵育20h后,基因缺失株出现较明显的抑菌圈,而野生株并未出现可见抑菌圈。3.免疫电镜对CB处理后基因缺失株及野生株形态观察各菌株与终浓度为0.5μg/m LCB孵育0.5h后,用纳米金标记的抗CB二抗与其作用后,免疫电镜可观察到完整的CB颗粒,野生株的细胞膜比基因缺失株光滑完整,且基因缺失株胞膜与胞质界限模糊,细胞破裂较多。在基因缺失株中,CB主要聚集在细菌胞膜上,而在野生株中,CB主要分布在细胞质中。4.Far Western-blot验证PSBP与CB互作将HAPS_2096基因克隆到原核表达载体上,成功获得可溶性融合蛋白GST-PSBP。与CB孵育后,利用抗CB抗体对其进行检测,Far Western-blot显示,CB与GST-PSBP有结合作用,而与GST标签对照蛋白无结合作用。以上结果表明,ABC型氨基酸转运泵的周质底物结合蛋白在副猪嗜血杆菌对CB抗性中起到十分重要的作用,其作用机制是通过与CB结合,可能将其转运到胞质内,减少其对细菌胞膜的破坏,从而表现出对CB的抗性。
[Abstract]:Haemophilus parasuis (Haemophilus parasuis HPS) is a common pathogen of pigs, pigs can cause Glaser S's disease (Gl sser? Disease), different age stages of pigs can be infected, many serotypes in clinic, not easy to control, and often mixed with other virus infection. In clinical treatment, this is the first choice of antibiotics for Disease Control and prevention, and the abuse of antibiotics will speed up the generation of antimicrobial resistance in Haemophilus influenzae side pig. Cecropin B (Cecropin B CB) is from hyalophora Cecropia found with natural broad-spectrum antimicrobial activity of insect antibacterial peptides (Antimicrobial, Peptide, AMP) is a potential treatment of infection the disease drug candidate, has great research value. In the previous study, we found that after CB induction, Haemophilus parasuis resistance, and some genes encoding gene products. HAPS_2096 Haemophilus parasuis ABC type amino Acid transporter periplasmic substrate binding protein (Periplasmic substrate binding protein, PSBP, ABC) transporter family is crucial in bacterial resistance mechanism. This study confirmed that HAPS_2096 plays an important role in Haemophilus parasuis resistance to CB, and to explore the mechanism of.1.HAPS_2096 gene mediated resistance gene mutant and deletion of complementary strains according to the homologous gene cross-over principle, construction has the same homology arm K-kan Dutch act P vector with HAPS_2096 gene, natural transformation HPS0135, obtained with a kanamycin resistance gene deletion strains. HAPS_2096 containing HAPS_2096 promoter and the whole open reading frame of the gene fragment was cloned into the vector of endogenous P SHk3 in the laboratory transformation on transformation to gene deletion strains, and transformed into the corresponding empty vector as control plants. The polarity effect, PCR, RT-PCR and Because the sequence of Blast verify the above strains were successfully constructed. The growth curve of the strain growth found no significant difference.2. strains of CB sensitivity test according to the CB of the HPS minimal inhibitory concentration (MIC) set up a series of CB concentration, the number of bacteria 105CFU/m L. gene deletion strains and wild strains CB and 1H, when CB the final concentration of 0.25 g/m L JS0135, the survival rate was 12.02%, significantly higher than that of a HAPS2096 survival rate was 5.16% (P0.05); similarly, when the final concentration of CB 0.125 g/m L JS0135, the survival rate was 26.62%, significantly higher than that of a HAPS2096 survival rate was 13.09% (P0.05) select the final concentration of 0.25 mu g/m L CB, 1H, under this concentration, containing the empty vector control. The survival rate was 1.81%, significantly lower than the complementary strain (P0.05). The survival rate was 6.82% in the agar diffusion test, CB solution 1 g/m L with agar hole, 37 DEG C after 20h incubation, gene deletion there was a bright line The bacteriostatic ring significantly, while the wild strains did not appear visible.3. immuno electron microscopy of bacteriostatic ring after treatment with CB gene deletion mutants and wild-type strains and the morphology of the final concentration of 0.5 g/m LCB after 0.5h incubation with nano gold labeled anti CB two and anti effect, can be observed after CB immunoelectron microscopy the whole particle, the cell membrane of wild strains than gene deletion strains were smooth and intact, and gene deletion strains of cell membrane and cytoplasm blurred, more cell rupture. In the mutant, CB mainly accumulates in the bacterial cell membrane, and in the wild strains, CB mainly distributed in the cytoplasm.4.Far Western-blot verification and PSBP the interaction of CB HAPS_2096 gene was cloned into the prokaryotic expression vector, successfully obtained the soluble fusion protein of GST-PSBP. and CB after incubation, detection of the use of anti CB antibody, Far Western-blot and GST-PSBP CB showed that the binding of GST, and according to the label free protein Binding. The above results suggest that the periplasmic substrate binding ABC amino acid transporter proteins in Haemophilus parasuis is very important for CB resistance plays, the mechanism is combined with the CB, may be transported to the cytoplasm, reduce the damage to the cell membrane, thus showing the resistance to CB.

【学位授予单位】:华中农业大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S852.61

【参考文献】

相关期刊论文 前1条

1 袁森泉 ,SimonOliveria,CarlosPijoan;副猪嗜血杆菌感染的诊断和防制策略[J];国外畜牧学(猪与禽);2003年02期



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