本文選題：副豬嗜血桿菌　切入點：天蠶素B　出處：《華中農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：副豬嗜血桿菌(Haemophilus parasuis,HPS)是豬的一種常見病原菌,可引起豬的格拉澤氏病(Gl?sser disease),不同年齡階段的豬均可感染,臨床上血清型眾多,不易控制,且常常與其他病毒混合感染。在臨床治療上,抗生素是該病防控的首選,而抗生素的濫用更加速了副豬嗜血桿菌耐藥性的產(chǎn)生。天蠶素B(Cecropin B,CB)是從惜古比天蠶中發(fā)現(xiàn)的具有廣譜抗菌活性的天然昆蟲抗菌肽(Antimicrobial Peptide,AMP),是一種潛在的治療感染性疾病的候選藥物,具有極大的研發(fā)價值。在前期的研究中,我們發(fā)現(xiàn)經(jīng)CB誘導(dǎo)后,副豬嗜血桿菌可產(chǎn)生耐藥性,并且與一些基因有關(guān)�；騂APS_2096編碼產(chǎn)物為副豬嗜血桿菌ABC型氨基酸轉(zhuǎn)運泵周質(zhì)底物結(jié)合蛋白(Periplasmic substrate binding protein,PSBP),ABC轉(zhuǎn)運泵家族在細菌耐藥機制中十分關(guān)鍵。本研究證實了HAPS_2096在副豬嗜血桿菌對CB的抗性中起到重要作用,并且試圖探索該基因介導(dǎo)耐藥的相關(guān)機制。1.HAPS_2096基因缺失株及缺失互補株的構(gòu)建根據(jù)同源基因交叉互換的原理,構(gòu)建具有與HAPS_2096基因有相同同源臂的自殺載體p K-kan,自然轉(zhuǎn)化HPS0135,獲得具有卡那霉素抗性的HAPS_2096基因缺失株。將含有HAPS_2096啟動子及其整個開放閱讀框的基因片段克隆到經(jīng)實驗室改造過的內(nèi)源載體p SHk3上,電轉(zhuǎn)化到基因缺失株中,同時轉(zhuǎn)化相應(yīng)空載體作為對照株。經(jīng)極性效應(yīng)、PCR、RT-PCR及基因序列Blast驗證上述各菌株構(gòu)建成功。測定生長曲線發(fā)現(xiàn)各菌株生長無明顯差異。2.各菌株對CB敏感性試驗根據(jù)CB對HPS最小抑菌濃度(MIC)設(shè)置一系列CB反應(yīng)濃度,細菌數(shù)量為105CFU/m L�；蛉笔е旰鸵吧昱cCB作用1h后,當CB終濃度為0.25μg/m L時,JS0135的存活率為12.02%,顯著高于ΔHAPS2096的存活率5.16%(p0.05);同樣,當CB終濃度為0.125μg/m L時,JS0135的存活率為26.62%,顯著高于ΔHAPS2096的存活率13.09%(p0.05)選擇終濃度0.25μg/m L的CB,作用1h后,在該濃度下,含空載體對照株存活率為1.81%,顯著低于互補株的存活率6.82%(p0.05)。在瓊脂擴散試驗中,將1μg/m L的CB溶液加入瓊脂孔中,37℃孵育20h后,基因缺失株出現(xiàn)較明顯的抑菌圈,而野生株并未出現(xiàn)可見抑菌圈。3.免疫電鏡對CB處理后基因缺失株及野生株形態(tài)觀察各菌株與終濃度為0.5μg/m LCB孵育0.5h后,用納米金標記的抗CB二抗與其作用后,免疫電鏡可觀察到完整的CB顆粒,野生株的細胞膜比基因缺失株光滑完整,且基因缺失株胞膜與胞質(zhì)界限模糊,細胞破裂較多。在基因缺失株中,CB主要聚集在細菌胞膜上,而在野生株中,CB主要分布在細胞質(zhì)中。4.Far Western-blot驗證PSBP與CB互作將HAPS_2096基因克隆到原核表達載體上,成功獲得可溶性融合蛋白GST-PSBP。與CB孵育后,利用抗CB抗體對其進行檢測,Far Western-blot顯示,CB與GST-PSBP有結(jié)合作用,而與GST標簽對照蛋白無結(jié)合作用。以上結(jié)果表明,ABC型氨基酸轉(zhuǎn)運泵的周質(zhì)底物結(jié)合蛋白在副豬嗜血桿菌對CB抗性中起到十分重要的作用,其作用機制是通過與CB結(jié)合,可能將其轉(zhuǎn)運到胞質(zhì)內(nèi),減少其對細菌胞膜的破壞,從而表現(xiàn)出對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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

【參考文獻】

相關(guān)期刊論文 前1條

1 袁森泉 ,SimonOliveria,CarlosPijoan;副豬嗜血桿菌感染的診斷和防制策略[J];國外畜牧學(豬與禽);2003年02期

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本文編號：1584244