支气管败血波氏杆菌Δhfq突变株的构建及生物学特性分析

发布时间：2018-03-22 15:57

  本文选题：支气管败血波氏杆菌　切入点：hfq基因　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：支气管败血波氏杆菌(Bordetella bronchiseptica,Bb),革兰氏阴性、需氧的球杆菌,感染可引发犬传染性气管支气管炎和兔波氏杆菌病,也是引起猪传染性萎缩性鼻炎的重要致病因子之一。Hfq参与调节细菌的多种生命活动,绿脓杆菌、大肠杆菌、鼠伤寒沙门氏菌、布氏杆菌、霍乱弧菌、单核细胞增生李斯特氏菌,脑膜炎奈瑟菌等hfq缺失后,细菌对小鼠的毒力明显减弱,对细胞的侵袭能力降低;同时针对其构建突变株,可获得减毒新型疫苗株。Hfq对支气管败血波氏杆菌的调节作用尚不清楚。因此,进行支气管败血波氏杆菌Δhfq突变株研究,明确Hfq的调节作用,可揭示支气管败血波氏杆菌的致病机制,也可为研究其减毒新型疫苗提供可能性。采用正向筛选同源重组技术,PCR扩增Bb122株hfq两端侧翼序列,将其克隆至质粒pBC-SK相应的酶切位点,构建自杀性质粒pBC-hfq;克隆卡那霉素抗性基因表达盒至hfq两端侧翼序列之间,构建重组转移质粒pBC-Δhfq。将pBC-Δhfq电转化至Bb122感受态细胞中。在抗性培养基上进行筛选,通过对卡那霉素抗性基因表达盒进行PCR和测序验证、对缺失的hfq进行PCR验证,表明卡那霉素抗性基因表达盒已经插入至hfq两端侧翼序列之间,hfq已经缺失完全,证明成功构建了支气管败血波氏杆菌Δhfq突变株。连续传20代,具有良好的遗传稳定性。体外生长曲线表明:在37℃培养时,Δhfq突变株在1~6 h生长速度基本和亲本菌株Bb122一致,6 h后生长速度略快于亲本菌株。进行抗逆性试验,在紫外线照射(UV)和50℃条件下,Δhfq突变株的耐受能力分别显著低于亲本菌株(p0.05);在H2O2条件下,Δhfq突变株与亲本菌株均无耐受能力。粘附和侵袭试验表明Δhfq突变株的粘附、侵袭能力低于亲本菌株,差异不显著。Δhfq突变株与亲本菌株分别对小鼠进行腹腔接种,Δhfq突变株以2.0×109 cfu剂量接种时,5只小鼠全部存活;亲本菌株以2.0×109 cfu剂量接种时,5只小鼠全部死亡,说明Δhfq突变株对小鼠的致病性是减弱的。小鼠免疫攻毒试验表明Δhfq突变株以2.0×109 cfu剂量免疫两次后,可耐受2.0×109 cfu剂量亲本菌株的攻击,说明Δhfq突变株具有良好的免疫原性。本研究构建了支气管败血波氏杆菌Δhfq突变株。Δhfq突变株具有良好的遗传稳定性和生长能力;Δhfq突变株对小鼠的毒力显著降低,初步明确hfq是与支气管败血波氏杆菌毒力相关的基因。Δhfq突变株具有良好的免疫原性,可为研究其减毒新型疫苗提供可能性,也可为Bb致病机制的研究奠定基础。
[Abstract]:Bordetella bronchiseptica, Gram-negative, aerobic Streptococcus spp., an infection that can cause infectious bronchitis in dogs and Bauhinella rabbit's disease. Hfq is also one of the important pathogenic factors causing infectious atrophic rhinitis in pigs. HFQ is involved in the regulation of various life activities of bacteria, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Brucella brucelis, Vibrio cholerae, Listeria monocytogenes. After hfq deletion of Neisseria meningitidis, the virulence of bacteria to mice was significantly decreased, and the ability of invasiveness to cells was decreased. The regulatory effect of attenuated new attenuated vaccine strain. HFQ on Bacillus bronchiae was not clear. Therefore, the study of 螖 hfq mutant strain of Bacillus bronchius was carried out to clarify the regulatory effect of Hfq. It can reveal the pathogenetic mechanism of Bacillus bronchus and provide the possibility for studying the novel attenuated vaccine. The flanking sequence of Bb122 strain hfq was amplified by positive screening homologous recombination technique and cloned to the corresponding restriction site of plasmid pBC-SK. The suicide plasmid pBC-hfqwas constructed, the kanamycin resistance gene expression box was cloned between the two flanking sequences of hfq, and the recombinant transfer plasmid pBC- 螖 hfq. pBC- 螖 hfq was electrotransformed into Bb122 receptive cells. The kanamycin resistant gene expression cassette was verified by PCR and sequencing, and the missing hfq was verified by PCR. The results showed that the kanamycin resistant gene expression box had been inserted into the flanking sequence of hfq. It is proved that the mutants 螖 hfq of Bacillus bronchus septicus were successfully constructed. The in vitro growth curve showed that the growth rate of 螖 hfq mutant was slightly faster than that of parent strain Bb122 after 6 h culture at 37 鈩，

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