共查询到18条相似文献，搜索用时 187 毫秒
1.
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
2.
应用15个VNTR位点对我国保存的19株布鲁菌标准株和4株减毒活疫苗以及松源地区10株临床分离株进行基因多态性研究。采用15个VNTR位点对33株布鲁菌进行PCR扩增,产物进行琼脂糖电泳,根据产物大小计算序列重复数,据此分析菌株的遗传多态性。建立了15个VNTR位点的琼脂糖凝胶电泳及拷贝数计算分析方法,应用该技术分析了33株布鲁菌的遗传多态性。结果表明这15个位点能较好地区分不同种型或亚型以及减毒活疫苗株,同时也为临床株的分型提供了依据。 相似文献
3.
为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。 相似文献
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布鲁菌是一种重要的人畜共患病病原,兼性胞内寄生,无典型毒力因子,毒力相关基因的研究对阐明布鲁菌持续感染的机制具有重要意义。本研究以流产布鲁菌S2308为亲本株,通过同源重组的方法,构建流产布鲁菌LysR家族转录调控子基因bab＿RS24670缺失株ΔlttR8,基于广宿主质粒pBBR1MCS构建成互补株ΔlttR8-Com。免疫印迹试验表明：ΔlttR8为光滑型菌株;体外培养分析发现lttR8缺失不影响布鲁菌的生长;细胞感染试验发现lttR8缺失不影响布鲁菌黏附、入侵细胞和胞内存活的能力。耐受性试验分析发现：lttR8缺失不影响布鲁菌对过氧化氢（H2O2）和多粘菌素B(polymyxin B)的敏感性。小鼠感染试验分析发现lttR8缺失显著减弱流产布鲁菌的毒力。综上所述,本研究发现一个新的与流产布鲁菌致病性相关的转录调控子,为布鲁菌致病机制研究提供参考。 相似文献
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首先采用RT-PCR鉴定布鲁菌16M在模拟胞内条件下BSR1955的转录,利用TargetRNA2预测BSR1955的靶基因;然后采用融合PCR构建BSR1955缺失株;将BSR1955插入pBBR1-MCS4质粒并电转入16M构建过表达株16M-BSR1955;分析缺失株和过表达株在模拟胞内环境和小鼠体内的存活能力。BSR1955在布鲁菌16M中存在转录且在不同刺激条件下转录水平不同;共预测出BSR1955的靶基因45个;BSR1955缺失或过表达影响布鲁菌16M在体外的生长能力,缺失BSR1955后在高盐、高渗环境中生存能力提高,第28和45天在小鼠脾脏内的细菌数量显著增加,表明BSR1955影响了布鲁菌的毒力。非编码小RNABSR1955影响布鲁菌16M在胞内的生存能力。 相似文献
6.
现有的布鲁菌减毒活疫苗存在一定毒力,且野强毒株和减毒活疫苗株间缺少可供鉴别的抗原,导致在血清学检测上自然感染与疫苗接种很难区分,限制了现有的减毒活疫苗的广泛应用.本文拟对布鲁菌的减毒活疫苗株S2进行遗传改造,克服上述缺陷.本研究利用同源重组的方法,得到了布鲁菌S2株omp10基因缺失株.分别用基因缺失株和疫苗株感染小鼠,比较基因缺失株小鼠体内的存活能力.结果成功构建了布鲁菌S2株omp10基因缺失株,动物试验结果表明,基因缺失株仍能在小鼠体内存活,具备作为减毒活疫苗的特性.与原始S2株比较,基因缺失株的感染力进一步减弱.表明omp10基因在布鲁菌的毒力及体内生存方面发挥了作用,为基因标记疫苗的研制奠定了基础. 相似文献
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《中国兽医学报》2016,(8):1358-1362
为找出布鲁菌疫苗株S2基因组分泌性蛋白、参与生物学途径基因、细胞组件基因、分子功能基因、毒力相关因子及耐药基因,对布鲁菌疫苗株S2全基因组测序并进行Ⅲ型分泌系统效应蛋白预测、GO注释、VFDB注释、ARDB注释。全基因组测序结果表明,S2基因组大小约为3.3 Mb,杂合度和重复度较低,GC含量分布未出现异常。通过Ⅲ型分泌系统效应蛋白预测未找出S2分泌性蛋白。通过GO数据库注释,发现参与生物学途径基因有3 823个、细胞组件基因有1 949个、分子功能基因有2 585个。通过VFDB数据库注释,找出S2基因组有79个毒力相关基因,其中最大基因为8 604bp,最小基因为180bp。通过ARDB数据库注释,找出S2基因组有13个耐药基因,最大基因为3 225bp,最小基因为333bp;其中功能注释的有GL002835基因,是枯草杆菌肽类基因,此基因耐受抗微生物类药物。本研究为进一步了解布鲁菌S2基因组基本功能奠定了基础。 相似文献
8.
应用代表性差异分析技术(Representational difference analysis,RDA)对流产布鲁菌强毒株544和疫苗株S2进行基因组差异分析。经过3轮差减杂交,对差异片段进行Southern-blot验证,BLAST分析和PCR鉴定,最终获得疫苗株S2特有的3条差异片段与已测序的所有猪布鲁菌(Brucella suis)同源性均为100%,只在弱毒株S2基因组中检测出,而在强毒株544中不存在,为建立布鲁菌自然感染菌与疫苗株的基因鉴别诊断方法奠定了基础。 相似文献
9.
布鲁菌属革兰氏阴性兼性胞内寄生菌,能感染多种宿主动物和人。该属可分为6个典型种,包括羊种、牛种、猪种、沙林鼠种、绵羊附睾种以及犬种布鲁菌等。此分类是基于其致病性以及宿主偏好性的差异划分。尽管6个种通过传统表型试验能区分,但布鲁菌种内采用DNA-DNA杂交证明DNA同源性高度一致（相似性大于90%）。因此有人提议布鲁菌由单一种组成,即布鲁菌属中只有羊种布鲁菌,其他种都是羊种菌的生物亚型之一。然而基于其他分子技术的基因分型表明其DNA多态性表现明显,说明目前对这个种的分型还是比较准确。而最近分离的海洋种布鲁氏菌分离株（鳍型和鲸型）采用传统分型标准和一些特异的分子标记也证明这种分型比较正确。本文对目前布鲁菌种属进化和分类学进行综述,希望对研究其进化和分类有所帮助。 相似文献
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A E Jensen N F Cheville C O Thoen A P MacMillan W G Miller 《Journal of veterinary diagnostic investigation》1999,11(2):152-157
Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species. 相似文献
12.
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
13.
Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3 总被引：3，自引：0，他引：3
One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars. 相似文献
14.
分子生物学技术在布鲁菌种型鉴定上的应用 总被引：1，自引：0，他引：1
由于布鲁菌种型较多、宿主广泛、传播途径多样,因此有必要对布鲁菌进行准确的种型鉴别,以利于该病的快速诊断、流行病学分析,并采取科学有效的防控措施。分子生物学技术的不断发展为布鲁菌种型鉴定提供了愈来愈丰富的手段,并逐渐成为布鲁菌遗传溯源研究的重要工具。MLVA和real-time PCR技术因具有重复性好、分辨率高等优势,成为布鲁菌种型鉴定研究关注的热点。多重PCR、RFLP、RAPD和REP等技术也在布鲁菌种型鉴定中得到广泛应用。为了解分子生物学技术在布鲁菌种型鉴定上的应用现状,对相关研究进行了综述。 相似文献
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16.
布氏杆菌为世界性具重要公共卫生意义的人兽共患疫病原，分6个种。建立种间及种株间安全敏感、经济有效的快速鉴别诊断方法对布病防制及分子流行病学研究具有重要意义。布氏杆菌IS711和omp2基因具有种属特异性，可用于布氏杆菌的PCR分子诊断。其中IS711为转座因子，在不同种布菌种存在插入位置的多态性，外膜蛋白OMP2编码基因则存在反向重复序列及种株间的多态性。为此，分别采用复式-PCR、PCR和限制性酶切片段长多态性（RFLP）分析，对分属于B．mclitcnsis、B．suis和B．abortus的不同种布氏杆菌的不同种株，M5、M16、S2、S6和S19进行分子鉴别诊断。结果显示，根据IS711基因特定PCR扩增片段长多态性，可进行布氏杆菌种间的快速鉴别；而omp2编码基因PCR扩增片段PsrⅠ、KpnⅠ、NcoⅠ和Eco47 Ⅲ等4种限制酶片段长多态性，则可成为布氏杆菌菌株间特异的分子鉴别诊断标记，甚至疫苗株M5和野毒株M16之间的分子诊断标记。 相似文献
17.
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
18.
Ferrão-Beck L Cardoso R Muñoz PM de Miguel MJ Albert D Ferreira AC Marín CM Thiébaud M Jacques I Grayon M Zygmunt MS Garin-Bastuji B Blasco JM Sá MI 《Veterinary microbiology》2006,115(1-3):269-277
Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method. 相似文献