表面蛋白EF0591對致羔羊腦炎糞腸球菌XJ05部分生物學特性影響的研究
發(fā)布時間：2018-09-09 13:08
【摘要】：腸球菌是一種條件致病菌,常棲居于人和動物消化道內(nèi),腸球菌不僅能引起人類感染,而且豬、牛、羊、禽、貓、犬等也是易感動物,并且能在人與動物之間水平傳播。當人體免疫力低下或腸球菌定植部位改變時,能引起人類疾病,如菌血癥、尿路感染、肝膽膿毒癥、心內(nèi)膜炎、外科傷口感染、牙髓感染、新生兒膿血癥等甚至危及生命。腸球菌的致病性與其毒力因子密切相關(guān),有研究表明,當腸球菌缺少某些毒力因子時,其毒力將大大降低。因此,研究腸球菌的毒力因子對研究腸球菌的致病機制具有重要的意義。EF0591是新近發(fā)現(xiàn)的一種毒力因子,有研究證明它與臨床分離株密切相關(guān),但是人們關(guān)于它毒力及作用機制還知之甚少。本研究以致羔羊腦炎糞腸球菌XJ05(E.faecalis XJ05)為親本株,構(gòu)建EF0591基因突變株(E.faecalis XJ05-△0591),培養(yǎng)He La及小鼠RAW264.7細胞,觀察突變該基因后對He La細胞的粘附能力、小鼠RAW264.7細胞的侵染能力、小鼠主要臟器的載菌能力以及對生物膜形成能力的影響。發(fā)現(xiàn)E.faecalis XJ05在細胞黏附、侵染能力方面都比E.faecalis XJ05-△0591強;E.faecalis XJ05在小鼠肝、心、腎、脾內(nèi)的載菌量均顯著多于E.faecalis XJ05-△0591,在腦中無明顯變化;E.faecalis XJ05生物膜的形成量較E.faecalis XJ05-△0591多,且形態(tài)結(jié)構(gòu)更加致密。主要研究內(nèi)容及結(jié)果如下:1、E.faecalis XJ05 EF0591基因突變株的構(gòu)建:應用同源重組技術(shù),利用PCR擴增出用于突變E.faecalis XJ05 EF0591基因片段,酶切、回收該基因片段,并與酶切的穿梭質(zhì)粒進行連接,電轉(zhuǎn)化至感受態(tài)的E.faecalis XJ05中。應用卡那霉素抗性篩選出陽性轉(zhuǎn)化子,并采用PCR等方法進行鑒定。最終成功得到基因突變株E.faecalis XJ05-△0591,經(jīng)過37℃連續(xù)傳代培養(yǎng)檢測證明該突變株具有良好的遺傳穩(wěn)定性。2、EF0591在E.faecalis XJ05的細胞黏附、侵染及小鼠臟器載菌量影響研究:向培養(yǎng)好的He La細胞中加入菌懸液孵育1h和2h,裂解細胞后進行計數(shù);向培養(yǎng)好的RAW264.7細胞中加入菌懸液,培養(yǎng)1h后加入雙抗,繼續(xù)培養(yǎng)3h和24h,裂解細胞后進行計數(shù);將制備好的菌懸液腹腔注射小鼠,在不同時間取小鼠,取肝臟、腎臟、脾臟、心臟和腦組織進行勻漿,勻漿液涂平板計數(shù)。以野毒株為對照,E.faecalis XJ05-△0591對He La細胞黏附數(shù)量下降,統(tǒng)計學比較差異顯著(P0.05)。對小鼠RAW264.7細胞的侵染數(shù)量下降,統(tǒng)計學比較差異顯著(P0.05)。E.faecalis XJ05-△0591在小鼠肝小葉、心臟、脾臟和腎臟中的載菌量下降,統(tǒng)計學比較差異顯著(P0.05);在腦組織中細菌數(shù)量有變化,但差異不顯著。3、EF0591對E.faecalis XJ05生物膜形成影響的研究:E.faecalis XJ05-△0591及E.faecalis XJ05親本株分別在相同條件下、于96孔板中、37℃恒溫箱中培養(yǎng),經(jīng)PBS清洗并干燥,結(jié)晶紫染色后測其不同時間點的OD595值。將突變株和野毒株置于放有細胞爬片的六孔板中,37℃恒溫箱中培養(yǎng),分別用結(jié)晶紫和FITC-Con A及PI染液染色,在光學顯微鏡和激光共聚焦顯微鏡下觀察不同時間點的生物膜。在不同時間所測得的OD595值顯示,E.faecalisXJ05-△0591形成的生物膜在6h形成明顯,24h達到最大,24h后逐漸開始降解,各個時間點的生物膜形成量都明顯低于E.faecalis XJ05親本株,且差異顯著(P0.05)。光學顯微鏡觀察E.faecalis XJ05-△0591及E.faecalis XJ05形成的生物膜形態(tài)發(fā)現(xiàn)E.faecalis XJ05形成的生物膜網(wǎng)狀結(jié)構(gòu)較多,形態(tài)較致密,膜內(nèi)細菌數(shù)也更多。激光共聚焦顯微鏡觀察發(fā)現(xiàn)E.faecalis XJ05與E.faecalis XJ05-△0591相比生物膜形成量更多,膜內(nèi)細菌數(shù)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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.611
【參考文獻】
相關(guān)期刊論文 前3條
1 柳建新;周霞;田晶華;朱翠萍;;綿羊人工感染糞腸球菌的病理組織學觀察[J];動物醫(yī)學進展;2008年01期
2 劉紅艷;韋曦;凌均h，
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