本文選題：支氣管敗血波氏桿菌　切入點(diǎn)：hfq基因　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：支氣管敗血波氏桿菌(Bordetella bronchiseptica,Bb),革蘭氏陰性、需氧的球桿菌,感染可引發(fā)犬傳染性氣管支氣管炎和兔波氏桿菌病,也是引起豬傳染性萎縮性鼻炎的重要致病因子之一。Hfq參與調(diào)節(jié)細(xì)菌的多種生命活動(dòng),綠膿桿菌、大腸桿菌、鼠傷寒沙門氏菌、布氏桿菌、霍亂弧菌、單核細(xì)胞增生李斯特氏菌,腦膜炎奈瑟菌等hfq缺失后,細(xì)菌對小鼠的毒力明顯減弱,對細(xì)胞的侵襲能力降低;同時(shí)針對其構(gòu)建突變株,可獲得減毒新型疫苗株。Hfq對支氣管敗血波氏桿菌的調(diào)節(jié)作用尚不清楚。因此,進(jìn)行支氣管敗血波氏桿菌Δhfq突變株研究,明確Hfq的調(diào)節(jié)作用,可揭示支氣管敗血波氏桿菌的致病機(jī)制,也可為研究其減毒新型疫苗提供可能性。采用正向篩選同源重組技術(shù),PCR擴(kuò)增Bb122株hfq兩端側(cè)翼序列,將其克隆至質(zhì)粒pBC-SK相應(yīng)的酶切位點(diǎn),構(gòu)建自殺性質(zhì)粒pBC-hfq;克隆卡那霉素抗性基因表達(dá)盒至hfq兩端側(cè)翼序列之間,構(gòu)建重組轉(zhuǎn)移質(zhì)粒pBC-Δhfq。將pBC-Δhfq電轉(zhuǎn)化至Bb122感受態(tài)細(xì)胞中。在抗性培養(yǎng)基上進(jìn)行篩選,通過對卡那霉素抗性基因表達(dá)盒進(jìn)行PCR和測序驗(yàn)證、對缺失的hfq進(jìn)行PCR驗(yàn)證,表明卡那霉素抗性基因表達(dá)盒已經(jīng)插入至hfq兩端側(cè)翼序列之間,hfq已經(jīng)缺失完全,證明成功構(gòu)建了支氣管敗血波氏桿菌Δhfq突變株。連續(xù)傳20代,具有良好的遺傳穩(wěn)定性。體外生長曲線表明:在37℃培養(yǎng)時(shí),Δhfq突變株在1~6 h生長速度基本和親本菌株Bb122一致,6 h后生長速度略快于親本菌株。進(jìn)行抗逆性試驗(yàn),在紫外線照射(UV)和50℃條件下,Δhfq突變株的耐受能力分別顯著低于親本菌株(p0.05);在H2O2條件下,Δhfq突變株與親本菌株均無耐受能力。粘附和侵襲試驗(yàn)表明Δhfq突變株的粘附、侵襲能力低于親本菌株,差異不顯著。Δhfq突變株與親本菌株分別對小鼠進(jìn)行腹腔接種,Δhfq突變株以2.0×109 cfu劑量接種時(shí),5只小鼠全部存活;親本菌株以2.0×109 cfu劑量接種時(shí),5只小鼠全部死亡,說明Δhfq突變株對小鼠的致病性是減弱的。小鼠免疫攻毒試驗(yàn)表明Δhfq突變株以2.0×109 cfu劑量免疫兩次后,可耐受2.0×109 cfu劑量親本菌株的攻擊,說明Δhfq突變株具有良好的免疫原性。本研究構(gòu)建了支氣管敗血波氏桿菌Δhfq突變株。Δhfq突變株具有良好的遺傳穩(wěn)定性和生長能力;Δhfq突變株對小鼠的毒力顯著降低,初步明確hfq是與支氣管敗血波氏桿菌毒力相關(guān)的基因。Δhfq突變株具有良好的免疫原性,可為研究其減毒新型疫苗提供可能性,也可為Bb致病機(jī)制的研究奠定基礎(chǔ)。
[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，

本文編號：1649349