stk基因对化脓隐秘杆菌耐药性和毒力影响的研究

发布时间：2018-01-01 10:23

本文关键词：stk基因对化脓隐秘杆菌耐药性和毒力影响的研究　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：真核样丝氨酸/苏氨酸激酶(eSTK)可通过自身磷酸化或对其它蛋白质进行磷酸化作用而对细菌的多种生理活动进行调节,包括生长发育、生物膜形成、耐药性和致病性的产生等。化脓隐秘杆菌是一种革兰氏阳性菌,能够引起猪、牛、羊等重要经济动物的化脓性感染。研究eSTK对化脓隐秘杆菌的耐药性和毒力的影响,将有助于揭示化脓隐秘杆菌耐药机制和致病机制,为化脓隐秘杆菌所致疾病的防治提供基础。通过对化脓隐秘杆菌临床分离菌株BM-H06-3全基因组序列的分析,确定了 eSTK的编码基因stk在全基因组DNA序列中的大小,并选择stk基因的上、下游同源臂,运用PCR扩增技术,以临床分离菌株的全基因组DNA为模板,PCR扩增上、下游同源臂,以pEASY-T1载体为模板,PCR扩增卡那抗性基因,构建重组质粒pBlue::stk。通过电转化法将重组质粒pBlue::stk转入化脓隐秘杆菌感受态,构建化脓隐秘杆菌stk缺失菌株,并采用PCR技术和测序等方法鉴定对stk缺失菌株;对比分析化脓隐秘杆菌stk基因缺失前后,在生长速率、溶血活性、药物敏感性和毒力方面的变化情况。化脓隐秘杆菌生长速率试验结果表明,与临床分离菌株相比,stk缺失菌株生长缓慢。采用肉汤微量稀释法测定化脓隐秘杆菌标准菌株、临床分离菌株和stk缺失菌株对常见11种抗生素的敏感性,结果显示,与临床分离菌株相比,头孢噻呋、青霉素和庆大霉素对stk缺失菌株的MIC值未发生变化,链霉素、阿米卡星、盐酸四环素和罗红霉素对stk缺失菌株的MIC值明显降低,说明stk缺失后导致化脓隐秘杆菌临床分离菌株对部分抗生素的敏感性升高。溶血活性试验结果表明,化脓隐秘杆菌stk基因缺失后溶血活性减弱。同时,临床分离菌株攻毒组的小鼠的存活率为25.0%,stk缺失菌株攻毒组小鼠的存活率为87.5%,表明化脓隐秘杆菌stk基因缺失后,细菌毒力下降。由此可知,化脓隐秘杆菌eSTK可能参与细菌细胞的生长、溶血活性、药物敏感性、细菌毒力的调节。本研究利用同源重组技术构建化脓隐秘杆菌stk缺失菌株,并对stk缺失菌株的生长速率、溶血活性、耐药性和毒力等进行研究,初步揭示了 eSTK对化脓隐秘杆菌的调节作用,将为化脓隐秘杆菌耐药机制和致病机理的深入研究提供思路,以期为临床有效预防和治疗化脓隐秘杆菌感染的研究奠定基础。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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