本文關(guān)鍵詞：stk基因?qū)撾[秘桿菌耐藥性和毒力影響的研究　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： stk 化膿隱秘桿菌 同源重組 耐藥性 毒力

【摘要】：真核樣絲氨酸/蘇氨酸激酶(eSTK)可通過(guò)自身磷酸化或?qū)ζ渌鞍踪|(zhì)進(jìn)行磷酸化作用而對(duì)細(xì)菌的多種生理活動(dòng)進(jìn)行調(diào)節(jié),包括生長(zhǎng)發(fā)育、生物膜形成、耐藥性和致病性的產(chǎn)生等�；撾[秘桿菌是一種革蘭氏陽(yáng)性菌,能夠引起豬、牛、羊等重要經(jīng)濟(jì)動(dòng)物的化膿性感染。研究eSTK對(duì)化膿隱秘桿菌的耐藥性和毒力的影響,將有助于揭示化膿隱秘桿菌耐藥機(jī)制和致病機(jī)制,為化膿隱秘桿菌所致疾病的防治提供基礎(chǔ)。通過(guò)對(duì)化膿隱秘桿菌臨床分離菌株BM-H06-3全基因組序列的分析,確定了 eSTK的編碼基因stk在全基因組DNA序列中的大小,并選擇stk基因的上、下游同源臂,運(yùn)用PCR擴(kuò)增技術(shù),以臨床分離菌株的全基因組DNA為模板,PCR擴(kuò)增上、下游同源臂,以pEASY-T1載體為模板,PCR擴(kuò)增卡那抗性基因,構(gòu)建重組質(zhì)粒pBlue::stk。通過(guò)電轉(zhuǎn)化法將重組質(zhì)粒pBlue::stk轉(zhuǎn)入化膿隱秘桿菌感受態(tài),構(gòu)建化膿隱秘桿菌stk缺失菌株,并采用PCR技術(shù)和測(cè)序等方法鑒定對(duì)stk缺失菌株;對(duì)比分析化膿隱秘桿菌stk基因缺失前后,在生長(zhǎng)速率、溶血活性、藥物敏感性和毒力方面的變化情況�；撾[秘桿菌生長(zhǎng)速率試驗(yàn)結(jié)果表明,與臨床分離菌株相比,stk缺失菌株生長(zhǎng)緩慢。采用肉湯微量稀釋法測(cè)定化膿隱秘桿菌標(biāo)準(zhǔn)菌株、臨床分離菌株和stk缺失菌株對(duì)常見(jiàn)11種抗生素的敏感性,結(jié)果顯示,與臨床分離菌株相比,頭孢噻呋、青霉素和慶大霉素對(duì)stk缺失菌株的MIC值未發(fā)生變化,鏈霉素、阿米卡星、鹽酸四環(huán)素和羅紅霉素對(duì)stk缺失菌株的MIC值明顯降低,說(shuō)明stk缺失后導(dǎo)致化膿隱秘桿菌臨床分離菌株對(duì)部分抗生素的敏感性升高。溶血活性試驗(yàn)結(jié)果表明,化膿隱秘桿菌stk基因缺失后溶血活性減弱。同時(shí),臨床分離菌株攻毒組的小鼠的存活率為25.0%,stk缺失菌株攻毒組小鼠的存活率為87.5%,表明化膿隱秘桿菌stk基因缺失后,細(xì)菌毒力下降。由此可知,化膿隱秘桿菌eSTK可能參與細(xì)菌細(xì)胞的生長(zhǎng)、溶血活性、藥物敏感性、細(xì)菌毒力的調(diào)節(jié)。本研究利用同源重組技術(shù)構(gòu)建化膿隱秘桿菌stk缺失菌株,并對(duì)stk缺失菌株的生長(zhǎng)速率、溶血活性、耐藥性和毒力等進(jìn)行研究,初步揭示了 eSTK對(duì)化膿隱秘桿菌的調(diào)節(jié)作用,將為化膿隱秘桿菌耐藥機(jī)制和致病機(jī)理的深入研究提供思路,以期為臨床有效預(yù)防和治療化膿隱秘桿菌感染的研究奠定基礎(chǔ)。
[Abstract]:Eukaryotic serine / threonine kinase (STK) can regulate many physiological activities of bacteria, including growth and development, biofilm formation, through self-phosphorylation or phosphorylation of other proteins. Drug resistance and pathogenicity, etc. Bacillus pyogenes is a Gram-positive bacteria that can cause pigs and cattle. Studies on the effect of eSTK on drug resistance and virulence of covert bacillus pyogenes will be helpful to reveal the mechanism of drug resistance and pathogenicity of S. pyogenes and other important economic animals. To provide the basis for the prevention and treatment of the diseases caused by the Bacillus pyogenes. The whole genome sequence of the clinical isolates BM-H06-3 was analyzed. The size of eSTK encoding gene stk in the whole genome DNA sequence was determined. The upstream and downstream arms of stk gene were selected and PCR amplification technique was used. The whole genome DNA of clinical isolates was used as template to amplify the kana-resistant gene. The downstream homologous arm was amplified by pEASY-T1 vector. The recombinant plasmid pBlue1: STK was constructed. The recombinant plasmid pBlue::stk was transformed into the susceptible state of Caetobacter pyogenes by electrotransformation, and the stk deletion strain was constructed. Stk deletion strains were identified by PCR and sequencing. The changes of growth rate, hemolytic activity, drug sensitivity and virulence before and after the deletion of stk gene were compared. Compared with the clinical isolates, the stk-deficient strains grew slowly. The standard strains of Stereobacterium pyogenes were determined by broth microdilution method. The sensitivity of clinical isolates and stk deficient strains to 11 common antibiotics showed that cefotaxime was more sensitive than clinical isolates. The MIC values of penicillin and gentamicin on stk deleted strains were not changed, but the MIC values of streptomycin, amikacin, tetracycline hydrochloride and roxithromycin on stk deleted strains were significantly decreased. The results of hemolytic activity test showed that the hemolytic activity of Stereobacterium purpura stk gene was decreased after the deletion of stk gene. The survival rate of mice in the clinical isolated strain attack group was 25.0% and the survival rate of the mice in the control group was 87.5%, indicating that the stk gene deletion of Stereobacterium purpurus was found. The decrease of bacterial virulence shows that eSTK may be involved in the growth, hemolytic activity and drug sensitivity of bacterial cells. The regulation of bacterial virulence. In this study, homologous recombination technique was used to construct the stk deletion strain, and the growth rate, hemolytic activity, drug resistance and virulence of the stk deficient strain were studied. This study revealed the regulatory effect of eSTK on Pseudomonas pyogenes, which will provide some ideas for the further study of drug resistance and pathogenicity of C. purpura. So as to lay a foundation for clinical study on prevention and treatment of cryptomegaly purpura infection.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.61

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本文編號(hào)：1364115