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1.
试验旨在构建布鲁氏菌Ⅳ型分泌系统vceA基因缺失株,分析其生长特征及其在人胚胎滋养层细胞(HPT-8)中的存活能力。以布鲁氏菌S2308为模板,PCR扩增vceA基因上、下游同源臂,以pUC19K质粒为模板扩增kan基因,然后利用融合PCR技术将3段基因进行融合,再将此片段与pMD19-T载体连接,电转至大肠杆菌DH5α感受态细胞中,构建ΔvceA基因缺失株(S2308ΔvceA),通过卡那抗性对缺失株进行连续筛选,并检测其遗传稳定性。在相同培养条件下,观察亲本株S2308和S2308ΔvceA的生长变化趋势,以侵染复数(MOI)100∶1侵染HPT-8细胞,通过CFU计数检测亲本株S2308和缺失株S2308ΔvceA在细胞内的生存能力。结果显示,试验成功获得片段大小为372、510、1 093 bp的vceA基因上、下游同源臂和kan基因;且成功构建pMD19-T-vceA-kan自杀载体,将其电转入大肠杆菌DH5α感受态细胞,构建基因缺失株,连续传代10次未发现基因回复突变;在体外相同培养条件下,缺失株S2308ΔvceA与亲本株S2308生长趋势相似,均在12 h达到对数生长期,30 h进入平台期;侵染HPT-8细胞12 h时,缺失株S2308ΔvceA在细胞中的数量显著低于亲本株S2308(P0.05)。综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌vceA基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在HPT-8细胞内的存活能力显著变弱。 相似文献
2.
【目的】 试验旨在构建牛种布鲁氏菌S2308的多铜氧化酶(BMCO)基因缺失株,探究缺失株的生长特性及在宿主细胞中的存活能力,并分析BMCO蛋白的结构。【方法】 利用PCR扩增BMCO基因上、下游同源臂和Kan基因,通过融合PCR技术将3段基因进行融合。融合片段与pMD19-T载体连接,制作牛布鲁氏菌S2308感受态细胞,1 800 V电压、400 Ω电阻电转化至牛种布鲁氏菌S2308感受态细胞中,涂布于Kan抗性的布鲁氏菌固体培养基中,筛选挑取阳性菌落,连续培养10代,检测第10代阳性菌落的遗传稳定性和生长变化趋势,将pBBR1MCS-4-BMCO融合质粒电转至能够稳定遗传BMCO基因缺失的牛种布鲁氏菌中,培养筛选阳性菌落。以感染复数(MOI)100分别用亲本株、缺失株、回补株侵染小鼠巨噬细胞RAW264.7,通过平板计数法分别检测3种菌株在细胞内的生存能力。【结果】 试验成功获得片段大小为522、539、1 054 bp的BMCO基因上、下游同源臂和Kan基因;成功构建pMD19-T-BMCO-Kan融合片段重组载体;获得稳定遗传的BMCO基因缺失株,命名为S2308ΔBMCO;成功构建BMCO基因回补株,命名为ΔBMCO::BMCOS2308;缺失株和亲本株表现的生长曲线相同,均在12 h达到对数生长期,30 h进入平台期;侵染小鼠巨噬细胞RWA264.7后,与亲本株S2308相比,S2308ΔBMCO株在胞内的生存能力极显著降低(P<0.01)。生物信息学分析结果表明,BMCO属于疏水蛋白,定位于胞质且含有大量的无规则卷曲、α-螺旋和延伸链,预示该蛋白具有多个结合位点。【结论】 本研究成功构建了布鲁氏菌BMCO基因的缺失株和回补株,BMCO基因的缺失不影响其生长性能,但其在宿主细胞内的存活能力显著性降低,初步分析了BMCO蛋白的结构,为后续布鲁氏菌分泌蛋白的功能研究奠定基础。 相似文献
3.
【目的】构建布鲁氏菌BPE159基因缺失株,研究缺失株体外生长变化特征及其在宿主细胞中的存活能力,探究布鲁氏菌感染期间分泌蛋白BPE159对自噬因子表达的影响。【方法】同源重组方法构建布鲁氏菌BPE159基因重组质粒,电转化布鲁氏菌S2308感受态细胞构建BPE159基因缺失株S2308ΔBPE159。PCR扩增BPE159基因,连接转化构建pBBR1MCS-4-BPE159载体,提取质粒进行电转化,构建BPE159基因回补株S2308ΔBPE159-C。琼脂糖凝胶电泳检测缺失株和回补株遗传稳定性。构建布鲁氏菌感染小鼠巨噬细胞RAW264.7模型,实时荧光定量PCR检测布鲁氏菌侵染后自噬细胞因子ATG5、Beclin1、LC3a和LC3b基因表达水平。以S2308、S2308ΔBPE159和S2308ΔBPE159-C株侵染小鼠巨噬细胞,收集细胞总RNA,实时荧光定量PCR检测BPE159基因缺失对布鲁氏菌侵染后自噬细胞因子表达水平的影响。在相同起始浓度下培养S2308、S2308ΔBPE159及S2308ΔBPE159-C株,观察细菌生长变化趋势;评价S2308ΔBPE159株在不同... 相似文献
4.
为了构建牛布鲁菌S2308(简称S2308)的铁转录调控因子rirA基因突变株(S2308ΔrirA),探讨该基因对自身生长的影响以及其对不同类型细胞黏附侵袭和胞内繁殖的作用。利用同源重组和抗性替换的方法,以卡那抗性基因替换rirA基因,获得突变株S2308ΔrirA。将亲本株S2308、疫苗株RB51和突变株S2308ΔrirA在相同营养条件下培养,观察其振荡培养时的生长变化趋势和静置培养时的聚集状态。将各菌株侵染人胚胎滋养层细胞(HPT-8)和小鼠巨噬细胞(RAW264.7),分别检测其黏附侵袭能力和胞内生存能力。结果显示,成功获得了布鲁菌rirA基因突变株且10代内未发生回复性突变;与亲本株相比,S2308ΔrirA在相同体外培养条件下其生长趋势未发生明显改变,且其凝集状态与亲本株类似;黏附侵袭试验显示,S2308ΔrirA对HPT-8细胞的黏附侵袭能力显著强于亲本株,而其对RAW264.7的黏附侵袭能力在一定程度上弱于亲本株;布鲁菌胞内生存试验发现,布鲁菌侵染HPT-8细胞24h后,其胞内细菌数量显著升高,而侵染巨噬细胞24h后,S2308ΔrirA的繁殖能力明显低于亲本株。这些结果表明,铁转录调控因子rirA基因参与了布鲁菌胞内寄生的过程,并与菌株的毒力强弱存在密切联系。 相似文献
5.
试验旨在分析糖基转移酶编码基因WadC影响布鲁氏菌胞内存活的作用。以羊种布鲁氏菌Rev.1基因组为模板,通过同源重组方法获得WadC基因上、下游同源臂融合片段,并与载体pUC19-SacB连接,构建pUC19-SacB-ΔwadC重组载体,电转至羊种布鲁氏菌Rev.1,构建ΔwadC缺失株（Rev.1ΔwadC）,检测菌株Rev.1ΔwadC的遗传稳定性,比较分析亲本株Rev.1和缺失株Rev.1ΔwadC的生长特性及其在BMDC和RAW264.7细胞中的生存能力。结果显示,试验成功构建基因缺失株,连续传代30次未发现基因回复突变;在体外相同培养条件下,缺失株Rev.1ΔwadC与亲本株Rev.1生长趋势相似,均在20 h到达对数生长期,44 h进入平台期;侵染BMDC细胞48和72 h时,其胞内存活率显著低于亲本株（P<0.05）;而侵染小鼠RAW264.7巨噬细胞试验显示,亲本菌株和基因缺失株无显著性差异（P>0.05）。综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌WadC基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在BMDC细胞内的存活能力显著变弱,为深入研究布鲁氏菌WadC基因功能奠定基础。 相似文献
6.
《中国预防兽医学报》2017,(10)
为获得毒力较弱且能够区分疫苗免疫与自然感染的羊种布鲁氏菌候选疫苗株,本研究构建羊种布鲁氏菌Rev.1疫苗株VirB12基因缺失突变株。分别扩增Rev.1疫苗株VirB12基因上下游同源臂序列以及卡那霉素抗性基因,采用融合PCR方法将3个基因片段连接构建突变盒,连接至pMD19-T载体,电转化入布鲁氏菌Rev.1感受态细胞筛选阳性克隆,获得Rev.1-ΔVirB12突变株。Rev.1-ΔVirB12连续传代15代未发生回复突变。羊种布鲁氏菌疫苗株Rev.1-ΔVirB12的构建为羊种布鲁氏菌疫苗研制奠定了基础。 相似文献
7.
《中国畜牧兽医》2020,(3)
为探究VirB基因对牛种布鲁氏菌A19株毒力的影响,深入了解布鲁氏菌胞内存活的机制,本研究以牛种布鲁氏菌A19株为模板,利用VirB基因的上下游同源臂融合kana抗性基因构建自杀质粒。以瞬间电击的方式,将自杀质粒电转进菌体,利用同源重组将VirB启动子用kana基因替换,构建A19ΔVirB缺失株。通过实时荧光定量PCR检测缺失株VirB相关蛋白的转录水平,并对缺失株的生长曲线、体外应激、黏附侵袭及胞内生存进行分析。结果显示,试验成功构建A19ΔVirB缺失株。实时荧光定量PCR结果显示,缺失株VirB相关蛋白的表达量极显著低于A19株(P0.01),其生长曲线虽然不同于亲本株,但生长趋势相同。体外应激结果显示,A19株和A19ΔVirB株热休克应激没有明显变化,但在强酸、强碱、高盐的刺激下,A19株的存活率显著高于A19ΔVirB株(P0.05)。黏附侵袭结果显示,缺失株对巨噬细胞的黏附侵袭能力近似于亲本株。胞内生存结果显示,随着时间的延长,A19株的胞内存活趋势逐渐上升,而缺失株A19ΔVirB总体趋势逐渐下降并且与A19的差距越来越大。综上所述,本研究成功构建了VirB启动子缺失株,极显著降低了相关蛋白的表达,为后续相关缺失株的构建及布鲁氏菌毒力的研究奠定基础。 相似文献
8.
探讨布鲁菌侵染人胚胎滋养层细胞(HPT-8)过程中对p38基因转录的影响。用牛布鲁菌2308和RB51株分别侵染HPT-8细胞,在不同侵染时间点(0、4、8、12、48h)收集细胞,提取总RNA,反转录获得cDNA;构建p38α、p38β和内参基因GAPDH的克隆质粒,并绘制3种基因扩增的标准曲线;RT-qPCR检测p38α和p38β基因的转录表达。结果显示,获得了HPT-8细胞侵染前后的cDNA,构建了pMD18-T-p38α/p38β/GAPDH克隆质粒,绘制了标准曲线。RT-qPCR结果显示,布鲁菌2308和RB51侵染滋养层细胞后(4、8、12、48h),p38α和p38β的相对表达量比0h均明显下降,差异极显著(P0.01),布鲁菌RB51株和2308株也存在一定差异。结果表明,在布鲁菌侵染HPT-8细胞的过程中,p38 MAPK信号通路被下调,提示p38 MAPK信号通路参与了对细胞生物学效应的调控,且这种调控作用与菌株的毒力强弱存在密切联系。 相似文献
9.
试验旨在探究ClpS基因在布鲁氏菌中的作用,分析比较ClpS基因突变对布鲁氏菌毒力的影响。利用同源重组技术,构建布鲁氏菌ClpS基因突变株,通过检测细菌生长曲线、细菌LPS合成能力及其在巨噬细胞内的存活能力和小鼠模型中的毒力,比较亲本株2308和突变株ΔClpS两者之间的差异。结果显示,在相同的培养条件下,亲本株2308和突变株ΔClpS的细菌浓度无明显差异,且两者提取的LPS银染结果基本一致,表明ClpS基因突变不影响布鲁氏菌生长速度,不影响细菌LPS合成;在细胞感染模型中,突变株ΔClpS在感染后72 h的胞内存活能力极显著低于亲本株2308（P<0.01）;小鼠感染试验显示,在感染后1周,亲本株2308感染组和突变株ΔClpS感染组小鼠脾脏重量及细菌含量无显著差异,但在感染后4周,突变株ΔClpS感染组的小鼠脾脏细菌含量为103.93 CFU/g脾脏,显著低于亲本株2308（106.68 CFU/g脾脏,P<0.01）,且突变株ΔClpS感染组的小鼠脾脏肿胀程度极显著低于亲本株2308（P<0.01）。综上所述,布鲁氏菌ClpS基因突变不影响细菌生长速度及细菌LPS合成能力,但ClpS基因突变可降低布鲁氏菌在小鼠脾脏内的定殖能力。 相似文献
10.
通过等位基因交换,分别敲除牛流产型布鲁菌减毒活疫苗S19株和缺失株S19-Δbp26的znuA基因,构建了布鲁菌znuA基因单缺失株S19-ΔznuA和bp26、znuA基因双缺失株S19-Δbp26-ΔznuA,对获得的2种缺失株进行形态学、生长特性、稳定性和基因测序验证。结果表明,S19株和S19-Δbp26株的znuA基因均成功缺失,生长特性表示2种缺失株与亲本株生长基本无差异;体外连续传至20代,菌落PCR鉴定及基因测序结果显示2种缺失株均遗传稳定。牛流产型布鲁菌单缺失株S19-ΔznuA和双缺失株S19-ΔznuA-Δbp26的成功构建为布鲁菌新型疫苗的研发、布鲁菌感染动物过程中ZnuA蛋白的作用机理及其与Bp26蛋白之间关系的研究奠定了基础。 相似文献
11.
Marianelli C La Rosa G Ciuchini F Muscillo M Pasquali P Adone R 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2003,50(10):494-499
DNA polymorphism of the alkB gene, a DNA repair gene, was assessed by PCR on Brucella abortus biovars 1 (strains 99, S19, 45/20, RB51 and 2308), 3 (Tulya strain), 5 (B3196 strain) and 6 (870 strain). A DNA repetitive element, named IS711, was detected in all studied biovars 1 and its complete nucleotide sequence was determined. We found that the element in alkB gene, bounded by 14 bp imperfect inverted repeats (IRs), is 840 bp long and appears to duplicate a consensus target site, CTAG. Analysing its nucleotide sequence of both forward and reverse strands, more than 10 open reading frames (ORFs) were found. Two potential transposase coding regions were chosen comparing all possible ORFs with the database. Comparing IS711 elements isolated from Brucella species, including both those characterized in our work and the published ones, differences in length and in nucleotide composition were observed among Brucella species, members of the same species and within the same strain. Our results confirm the heterogeneity of IS711 elements in Brucella genus and suggest the possibility to use this element to assess gene and genome diversity and to identify new molecular markers for Brucella species. 相似文献
12.
LIU Yufu DONG Hao SUN Shijing PENG Xiaowei CHEN Ruiai DING Jiabo JIANG Hui 《中国畜牧兽医》2007,47(11):3767-3773
The present study aimed to determine the role of ClpS gene,and to analyse the impact of ClpS mutation on the virulence of Brucella.A ClpS gene mutant strain,named ΔClpS was constructed by homologous recombination technology.The bacterial growth kinetics,the LPS synthesis ability and the survival ability of bacterial within macrophages as well as the virulence in mouse model were measured.In addition,the difference between parent strain 2308 and the mutant strain ΔClpS were compared.The results showed that under the same culture conditions,no difference in bacterial concentration was observed between 2308 and ΔClpS strains.The silver staining examination showed that the expression level of LPS extracted from two strains were similar,indicating ClpS gene mutation did not alter the growth rate and LPS synthesis ability of Brucella. In the cell infection assay,the survival ability of ΔClpS strain in cells was extremely significantly lower than that of 2308 strain at 72 h after infection (P<0.01).The results of mouse infection experiment showed that in the first week after infection,no significant difference in spleen weight and bacterial concentration between 2308 and ΔClpS strains infected mice was observed.However,at 4 weeks after infection,the bacterial concentration in spleen of ΔClpS infected mice was 103.93 CFU/g spleen,which was significantly lower than that of 2308 strain (106.68 CFU/g spleen,P<0.01).The spleen weight of ΔClpS infected mice was also remarkably lower than that of 2308 strain (P<0.01).In summary,the results suggested that the ClpS gene of Brucella did not play a role in Brucella growth rate and ability of LPS synthesis,whereas ClpS gene mutation decreased the ability of Brucella colonization in mouse spleen. 相似文献
13.
旨在探讨STAT6介导的巨噬细胞极化对布鲁氏菌胞内生存的影响。本研究采用布鲁氏菌光滑株S2308（S2308）和粗糙型疫苗株RB51（RB51）侵染巨噬细胞。利用qRT-PCR检测M1型巨噬细胞标志因子p65、NOS2和IL-1β,M2型巨噬细胞标志因子STAT6、ARG1、IL-10的mRNA表达水平;流式细胞术检测M1型标记分子CD86和M2型标记分子CD206的表达;Western blot检测p-STAT6蛋白及抑制剂AS对蛋白的抑制作用;ELISA检测M1型细胞因子TNF-α、IL-12和M2型细胞因子IL-4、IL-10的表达量;最后对胞内菌落进行CFU计数。qRT-PCR结果显示,在感染8、12 h时可显著诱导M1型因子mRNA转录表达,72 h时低表达,而M2型因子在72 h时高表达;流式细胞术结果显示,S2308感染12 h可显著诱导CD86的表达,感染72 h可显著诱导CD206的表达,但RB51对二者无影响;Western blot结果显示,S2308菌株在感染72 h时激活STAT6信号通路,而RB51几乎不激活该通路,抑制剂AS在2 μmol·L-1浓度时抑制效果最佳;ELISA结果显示,AS抑制剂可显著抑制IL-4、IL-10的释放,并促进TNF-α、IL-12的释放;CFU计数结果显示,S2308组的胞内菌呈先降低后显著上升趋势,加入AS抑制剂后可显著抑制布鲁氏菌胞内复制。布鲁氏菌S2308在感染后期能够通过STAT6诱导M1型巨噬细胞向M2型转化,并促进Th2型细胞因子的释放,从而有利于布鲁氏菌的胞内生存。而RB51几乎不激活该通路,不影响胞内生存。 相似文献
14.
8株布氏杆菌BCSP31基因的序列分析 总被引：4，自引：0，他引：4
参照GenBank中布氏杆菌的全基因组序列，设计一对特异性引物，通过PCR方法，扩增了8株来自3个布氏杆菌种的布氏杆菌表面蛋白31（BCSP31）全基因。PCR产物经克隆和序列分析后发现，该基因非常保守，8株不同菌株间的同源性高于99．3％；进化关系分析显示，5株中国来源的菌株（S2，M5，M111，M28，A387）显示了最近的同源关系，其中2个弱毒菌株S2和M5的BCSP31基因序列100％同源。3株外国来源的菌株（S19，2308，Rev．1）中，S19和2308同源性为100％。Rev．1与其他7株布氏杆菌BCSP31基因均有较大的差异。序列分析结果表明，BCSP31基因序列差异与布氏杆菌的种属和毒力无相关性。 相似文献
15.
The purpose of the test was to analyze the role of the glycosyltransferase-encoding gene WadC in affecting the intracellular survival of Brucella.Using the Brucella sheep Rev.1 genome as template,the fusion fragments of the homologous arms of the upper and lower arms of WadC gene were obtained by homologous recombination,and ligated to the vector pUC19-SacB to construct the pUC19-SacB-ΔwadC recombinant vector,which was transferred to sheep species Brucella Rev.1,constructing a ΔwadC deletion strain (Rev.1ΔwadC),testing the genetic stability of the strain Rev.1ΔwadC,comparing and analyzing the growth characteristics of the parental strain Rev.1 and the deletion strain Rev.1ΔwadC and the BMDC and RAW264.7 viability of cells.The results showed that the gene-deficient strain was successfully constructed in the experiment,and no genetic back mutation was found in 30 consecutive passages.Under the same culture conditions in vitro,the growth trend of the deleted strain Rev.1ΔwadC was similar to that of the parental strain Rev.1,and both reached logarithmic growth period at 20 h and reached plateau period at 44 h.When the BMDC cells were infected at 48 and 72 h,the intracellular survival rate was significantly lower than that of the parent strain (P<0.05).The RAW264.7 macrophage test of infected mice showed that the parent strain had no significant difference with the gene deletion strain (P>0.05).To sum up,this experiment successfully constructed and obtained a strain of Brucella WadC gene with good genetic stability.The deletion strain had similar growth trend with the parent strain under in vitro culture conditions;However,the survival ability of the deletion strain in BMDC cells was significantly weakened.This study laid a foundation for further study on the function of WadC gene of Brucella. 相似文献
16.
Adone R Ciuchini F La Rosa G Marianelli C Muscillo M 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2001,48(2):107-113
Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains. 相似文献