表面蛋白EF0591对致羔羊脑炎粪肠球菌XJ05部分生物学特性影响的研究
发布时间：2018-09-09 13:08
【摘要】：肠球菌是一种条件致病菌,常栖居于人和动物消化道内,肠球菌不仅能引起人类感染,而且猪、牛、羊、禽、猫、犬等也是易感动物,并且能在人与动物之间水平传播。当人体免疫力低下或肠球菌定植部位改变时,能引起人类疾病,如菌血症、尿路感染、肝胆脓毒症、心内膜炎、外科伤口感染、牙髓感染、新生儿脓血症等甚至危及生命。肠球菌的致病性与其毒力因子密切相关,有研究表明,当肠球菌缺少某些毒力因子时,其毒力将大大降低。因此,研究肠球菌的毒力因子对研究肠球菌的致病机制具有重要的意义。EF0591是新近发现的一种毒力因子,有研究证明它与临床分离株密切相关,但是人们关于它毒力及作用机制还知之甚少。本研究以致羔羊脑炎粪肠球菌XJ05(E.faecalis XJ05)为亲本株,构建EF0591基因突变株(E.faecalis XJ05-△0591),培养He La及小鼠RAW264.7细胞,观察突变该基因后对He La细胞的粘附能力、小鼠RAW264.7细胞的侵染能力、小鼠主要脏器的载菌能力以及对生物膜形成能力的影响。发现E.faecalis XJ05在细胞黏附、侵染能力方面都比E.faecalis XJ05-△0591强;E.faecalis XJ05在小鼠肝、心、肾、脾内的载菌量均显著多于E.faecalis XJ05-△0591,在脑中无明显变化;E.faecalis XJ05生物膜的形成量较E.faecalis XJ05-△0591多,且形态结构更加致密。主要研究内容及结果如下:1、E.faecalis XJ05 EF0591基因突变株的构建:应用同源重组技术,利用PCR扩增出用于突变E.faecalis XJ05 EF0591基因片段,酶切、回收该基因片段,并与酶切的穿梭质粒进行连接,电转化至感受态的E.faecalis XJ05中。应用卡那霉素抗性筛选出阳性转化子,并采用PCR等方法进行鉴定。最终成功得到基因突变株E.faecalis XJ05-△0591,经过37℃连续传代培养检测证明该突变株具有良好的遗传稳定性。2、EF0591在E.faecalis XJ05的细胞黏附、侵染及小鼠脏器载菌量影响研究:向培养好的He La细胞中加入菌悬液孵育1h和2h,裂解细胞后进行计数;向培养好的RAW264.7细胞中加入菌悬液,培养1h后加入双抗,继续培养3h和24h,裂解细胞后进行计数;将制备好的菌悬液腹腔注射小鼠,在不同时间取小鼠,取肝脏、肾脏、脾脏、心脏和脑组织进行匀浆,匀浆液涂平板计数。以野毒株为对照,E.faecalis XJ05-△0591对He La细胞黏附数量下降,统计学比较差异显著(P0.05)。对小鼠RAW264.7细胞的侵染数量下降,统计学比较差异显著(P0.05)。E.faecalis XJ05-△0591在小鼠肝小叶、心脏、脾脏和肾脏中的载菌量下降,统计学比较差异显著(P0.05);在脑组织中细菌数量有变化,但差异不显著。3、EF0591对E.faecalis XJ05生物膜形成影响的研究:E.faecalis XJ05-△0591及E.faecalis XJ05亲本株分别在相同条件下、于96孔板中、37℃恒温箱中培养,经PBS清洗并干燥,结晶紫染色后测其不同时间点的OD595值。将突变株和野毒株置于放有细胞爬片的六孔板中,37℃恒温箱中培养,分别用结晶紫和FITC-Con A及PI染液染色,在光学显微镜和激光共聚焦显微镜下观察不同时间点的生物膜。在不同时间所测得的OD595值显示,E.faecalisXJ05-△0591形成的生物膜在6h形成明显,24h达到最大,24h后逐渐开始降解,各个时间点的生物膜形成量都明显低于E.faecalis XJ05亲本株,且差异显著(P0.05)。光学显微镜观察E.faecalis XJ05-△0591及E.faecalis XJ05形成的生物膜形态发现E.faecalis XJ05形成的生物膜网状结构较多,形态较致密,膜内细菌数也更多。激光共聚焦显微镜观察发现E.faecalis XJ05与E.faecalis XJ05-△0591相比生物膜形成量更多,膜内细菌数更多。
[Abstract]:Enterococci are opportunistic pathogens, often inhabiting the digestive tract of humans and animals. Enterococci can not only cause human infection, but also * pigs, cattle, sheep, birds, cats, dogs and so on are also susceptible animals, and can be transmitted horizontally between humans and animals. When human immunity is low or the colonization site of Enterococcus is changed, it can cause human diseases, such as bacteremia and urine. Road infections, hepatobiliary sepsis, endocarditis, surgical wound infections, pulp infections, neonatal sepsis, and even life-threatening. Enterococcal pathogenicity is closely related to its virulence factors. Studies have shown that when Enterococcus lacks certain virulence factors, its virulence will be greatly reduced. Therefore, the study of enterococcal virulence factors for the study of Enterococcus EF0591 is a recently discovered virulence factor, which has been proved to be closely related to clinical isolates. However, little is known about its virulence and mechanism of action. XJ05-delta 0591) was used to culture He La and RAW264.7 cells of mice. The adhesion ability of the mutant gene to He La cells, the infection ability of RAW264.7 cells, the ability of carrying bacteria in the main organs of mice and the effect of biofilm formation were observed. It was found that E.faecalis XJ05 was stronger than E.faecalis XJ05-delta 0591 in cell adhesion and infection ability. E. faecalis XJ05 had more bacteria in liver, heart, kidney and spleen than E. faecalis XJ05 - Delta 0591, and had no significant change in brain; E. faecalis XJ05 had more biofilm formation than E. faecalis XJ05 - Delta 0591, and its morphology was more compact. The gene fragment of E.faecalis XJ05 EF0591 was amplified by homologous recombination using PCR. The fragment was digested by enzyme and recovered. The fragment was linked with the enzyme digested shuttle plasmid and electrotransformed into the competent E.faecalis XJ05. Positive transformants were screened by kanamycin resistance and identified by PCR. The mutant E. faecalis XJ05 - Delta 0591 showed good genetic stability after continuous subculture at 37. 2. EF0591 adhered to E. faecalis XJ05 cells, and the effect of infection and the amount of bacteria in the organs of mice was studied. The cultured RAW264.7 cells were added with bacterial suspension and then cultured for 1 hour with double antibodies. The cells were cultured for 3 and 24 hours and then lysed for counting. The amount of bacteria in liver lobule, heart, spleen and kidney of mice decreased significantly (P The effect of EF0591 on the biofilm formation of E. faecalis XJ05: E. faecalis XJ05 - Delta 0591 and E. faecalis XJ05 parental strains were cultured in 96-well plate, 37 C thermostat, washed and dried by PBS, and their OD595 values at different time points were measured after crystal violet staining. The biofilms of E. faecalis XJ05 - Delta 0591 were observed under optical microscope and laser confocal microscope at different time points. The OD595 values measured at different time points showed that the biofilms formed by E. faecalis XJ05 - Delta 0591 were in the shape of 6h. The amount of biofilm formation at each time point was significantly lower than that of E. faecalis XJ05 parental strain (P 0.05). The morphology of biofilm formed by E. faecalis XJ05 - Delta 0591 and E. faecalis XJ05 was observed by optical microscope. The results showed that E. faecalis XJ05 formed more biofilm network structure and shape. Laser confocal microscopy showed that E. faecalis XJ05 and E. faecalis XJ05 - Delta 0591 produced more biofilms and more bacteria than E. faecalis XJ05 - Delta 0591.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611
[Abstract]:Enterococci are opportunistic pathogens, often inhabiting the digestive tract of humans and animals. Enterococci can not only cause human infection, but also * pigs, cattle, sheep, birds, cats, dogs and so on are also susceptible animals, and can be transmitted horizontally between humans and animals. When human immunity is low or the colonization site of Enterococcus is changed, it can cause human diseases, such as bacteremia and urine. Road infections, hepatobiliary sepsis, endocarditis, surgical wound infections, pulp infections, neonatal sepsis, and even life-threatening. Enterococcal pathogenicity is closely related to its virulence factors. Studies have shown that when Enterococcus lacks certain virulence factors, its virulence will be greatly reduced. Therefore, the study of enterococcal virulence factors for the study of Enterococcus EF0591 is a recently discovered virulence factor, which has been proved to be closely related to clinical isolates. However, little is known about its virulence and mechanism of action. XJ05-delta 0591) was used to culture He La and RAW264.7 cells of mice. The adhesion ability of the mutant gene to He La cells, the infection ability of RAW264.7 cells, the ability of carrying bacteria in the main organs of mice and the effect of biofilm formation were observed. It was found that E.faecalis XJ05 was stronger than E.faecalis XJ05-delta 0591 in cell adhesion and infection ability. E. faecalis XJ05 had more bacteria in liver, heart, kidney and spleen than E. faecalis XJ05 - Delta 0591, and had no significant change in brain; E. faecalis XJ05 had more biofilm formation than E. faecalis XJ05 - Delta 0591, and its morphology was more compact. The gene fragment of E.faecalis XJ05 EF0591 was amplified by homologous recombination using PCR. The fragment was digested by enzyme and recovered. The fragment was linked with the enzyme digested shuttle plasmid and electrotransformed into the competent E.faecalis XJ05. Positive transformants were screened by kanamycin resistance and identified by PCR. The mutant E. faecalis XJ05 - Delta 0591 showed good genetic stability after continuous subculture at 37. 2. EF0591 adhered to E. faecalis XJ05 cells, and the effect of infection and the amount of bacteria in the organs of mice was studied. The cultured RAW264.7 cells were added with bacterial suspension and then cultured for 1 hour with double antibodies. The cells were cultured for 3 and 24 hours and then lysed for counting. The amount of bacteria in liver lobule, heart, spleen and kidney of mice decreased significantly (P The effect of EF0591 on the biofilm formation of E. faecalis XJ05: E. faecalis XJ05 - Delta 0591 and E. faecalis XJ05 parental strains were cultured in 96-well plate, 37 C thermostat, washed and dried by PBS, and their OD595 values at different time points were measured after crystal violet staining. The biofilms of E. faecalis XJ05 - Delta 0591 were observed under optical microscope and laser confocal microscope at different time points. The OD595 values measured at different time points showed that the biofilms formed by E. faecalis XJ05 - Delta 0591 were in the shape of 6h. The amount of biofilm formation at each time point was significantly lower than that of E. faecalis XJ05 parental strain (P 0.05). The morphology of biofilm formed by E. faecalis XJ05 - Delta 0591 and E. faecalis XJ05 was observed by optical microscope. The results showed that E. faecalis XJ05 formed more biofilm network structure and shape. Laser confocal microscopy showed that E. faecalis XJ05 and E. faecalis XJ05 - Delta 0591 produced more biofilms and more bacteria than E. faecalis XJ05 - Delta 0591.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611
【参考文献】
相关期刊论文 前3条
1 柳建新;周霞;田晶华;朱翠萍;;绵羊人工感染粪肠球菌的病理组织学观察[J];动物医学进展;2008年01期
2 刘红艳;韦曦;凌均h，
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