Brucella evolution and taxonomy

The genus Brucella contains alpha-Proteobacteria adapted to intracellular life within cells of a variety of mammals. Controversy has arisen concerning Brucella internal taxonomy, and it has been proposed that the DNA–DNA hybridization-based genomospecies concept be applied to the genus. According to this view, only one species, Brucella melitensis, should be recognized, and the classical species should be considered as biovars (B. melitensis biovar melitensis; B. melitensis biovar abortus; etc.). However, a critical reappraisal of the species concept, a review of the population structure of bacteria and the analysis of Brucella genetic diversity by methods other than DNA–DNA hybridization show that there are no scientific grounds to apply the genomospecies concept to this genus. On the other hand, an enlarged biological species concept allows the definition of Brucella species that are consistent with molecular analyses and support the taxonomical standing of most classical species. Both the host range as a long-recognized biological criterion and the presence of species-specific markers in outer membrane protein genes and in other genes show that B. melitensis, B. abortus, B. ovis, B. canis and B. neotomae are not mere pathovars (or nomenspecies) but biologically meaningful species. The status of B. suis is, however, less clear. These approaches should be useful to define species for the marine mammal Brucella isolates, as illustrated by the grouping of the isolates from pinnipeds or from cetaceans by omp2 gene analysis. It is shown that a correct Brucella species definition is important to understand the evolution of the genus.     

PCR as a diagnostic tool for brucellosis

Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of human brucellosis or contamination of food products). In these cases, a genus-specific PCR assay is sufficient. Genus-specific assays tend to be simple, robust, and somewhat permissive of environmental influences. The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S–23S rRNA operon.

Other instances require identification of the Brucella species involved. For example, most government-sponsored brucellosis eradication programs include regulations that stipulate a species-specific response. For epidemiological trace back, strain-specific identification is helpful. Typically, differential PCR-based assays tend to be more complex and consequently more difficult to perform. Several strategies have been explored to differentiate among Brucella species and strains, including locus specific multiplexing (e.g. AMOS-PCR based on IS711), PCR-RFLP (e.g. the omp2 locus), arbitrary-primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in molecular diagnostics for Brucella including the development of procedures designed for the direct analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for improvement.     

Molecular and cellular interactions between Brucella abortus antigens and host immune responses

Host protection against Brucella abortus, is thought to be mediated primarily by a Th1 type immune response. Unfortunately, only few specific bacterial antigens involved in stimulating protective cellular immunity against Brucella are known. Therefore, identifying bacterial proteins that induce a T-lymphocyte mediated response is critical to determine Brucella immunity. Several library screening methods are discussed that have been used to identify Brucella proteins that stimulate T lymphocytes including cellular immunoblotting, Escherichia coli expressed Brucella proteins, green fluorescence reporter systems, and signature tagged mutagenesis. Future studies would likely examine how bacterial proteins expressed within host cells aid pathogen survival and/or induce host responses. Some of these newly identified bacterial gene products may serve as antigens to activate a protective host immune response. Also, identifying Brucella proteins expressed at particular times during infection will also yield insights into Brucella pathogenesis.     

石河子地区宠物犬蜱的种类鉴定及其携带布鲁氏菌的检测

为了解新疆石河子地区宠物犬体表寄生蜱的种类及蜱携带布鲁氏菌情况,本试验在形态学分类的基础上,基于线粒体基因12S rRNA和细胞色素氧化酶Ⅰ（COⅠ）对宠物犬体表采集寄生蜱进行分子生物学检测,使用DNAMAN软件比对分析序列同源性,并应用序列分析软件Mega 7.0邻接法构建蜱种系统发育进化树,分析蜱种的遗传进化关系;基于布鲁氏菌外膜蛋白Omp22基因对宠物犬体表寄生蜱进行布鲁氏菌PCR检测,确定宠物犬蜱布鲁氏菌携带情况。结果显示,宠物犬体表寄生的436只蜱的形态学与线粒体基因（12S rRNA和COⅠ）分子生物学鉴定结果一致,均为新疆优势蜱种之一——图兰扇头蜱（Rhipicephalus turanicus）。基于12S rRNA基因构建的蜱种系统发育树显示,本试验所得宠物犬体表寄生图兰扇头蜱序列与GenBank中已知图兰扇头蜱的序列聚为一大支。基于布鲁氏菌Omp22基因的巢式PCR扩增结果显示,宠物犬体表寄生图兰扇头蜱携带布鲁氏菌的阳性率为4.82%（21/436）,且同源性为100%。BLAST比对显示,本试验所得宠物犬图兰扇头蜱携带的布鲁氏菌与新疆流行株YC31（GenBank登录号：MK201679.1）、QH5（GenBank登录号：MK201678.1）、EM3（GenBank登录号：MK201677.1）和ML9（GenBank登录号：MK201676.1）的同源性均为100%。本研究通过形态学及分子生物学探讨了新疆石河子地区宠物犬体表寄生蜱的种类及携带布鲁氏菌基本情况,为宠物犬体表寄生蜱的种类及蜱传疾病的监测和控制等研究工作提供了基础资料。     

NF-κB信号通路调控布鲁氏菌胞内存活分子机制的初步研究

本试验用布鲁氏菌强、弱毒株侵染小鼠巨噬细胞RAW264.7,旨在探讨NF-κB信号通路与布鲁氏菌强、弱毒株在胞内生存的关系。采用光滑型牛布鲁氏菌2308、粗糙型牛布鲁氏菌RB51在不同感染复数下侵染小鼠巨噬细胞RAW264.7,侵染0、4、8、24 h后,裂解细胞收集蛋白,Western blotting检测布鲁氏菌对激活NF-κB信号通路的影响。利用不同浓度的NF-κB信号通路抑制剂处理小鼠巨噬细胞RAW264.7,然后用布鲁氏菌在不同感染复数下侵染小鼠巨噬细胞RAW264.7,ELISA试剂盒检测细胞因子TNF-α、IL-1β、IL-6的表达量;同时对胞内菌CFU进行计数。结果显示粗糙型牛布鲁氏菌RB51可以强烈激活NF-κB信号通路,光滑型牛布鲁氏菌2308对其激活作用较弱;同时对NF-κB信号通路的激活具有浓度依赖性,在感染复数为80:1、侵染时间为8 h时光滑型牛布鲁氏菌2308和粗糙型牛布鲁氏菌RB51对NF-κB激活程度最强,且该通路参与产生TNF-α、IL-1β和IL-6;NF-κB信号通路抑制剂BAY11-7082影响布鲁氏菌在胞内的生存。因此,粗糙型牛布鲁氏菌RB51胞内存活与NF-κB信号通路密切相关,为进一步研究布鲁氏菌的胞内致病机制奠定基础,也为布鲁氏菌新型药物的研发、家畜布鲁氏菌病预防和治疗提供科学依据。     

Preliminary Study on NF-κB Signaling Pathway Regulating Brucella Intracellular Survival Molecular Mechanisms

By the infection of Brucella virulent strain and attenuated strain in mice macrophage RAW264.7,the assay was aimed to explore the relationship between NF-κB signaling pathways and Brucella virulent strain and attenuated strain in intracellular survival.Use different MOI Brucella (2308,RB51,16M and M5) to infect mice macrophage RAW264.7,after 0,4,8 and 24 h infected,cracking cell and collecting supernatant,we detected the effect of Brucella on activation of NF-κB signaling pathway by Western blotting.Different concentrations of NF-κB signaling pathway inhibitor were incubated with mice macrophage RAW264.7,with different multiplicities of infection (MOI) of Brucella infecting cells,ELISA kits to detect the expressions of TNF-α,IL-1β and IL-6 cytokine;At the same time,count the number of intracellular bacteria of CFU.The results showed that rough cattle Brucella strains RB51 could strongly activate NF-κB signaling pathway,smooth cattle Brucella strains 2308 was weak in the activation;At the same time,the activation of NF-κB signaling pathway was concentration dependent.When the MOI was 80,infection time was 8 h,NF-κB activation degrees of rough cattle Brucella strains RB51 and smooth cattle Brucella strains 2308 were the strongest,and this pathway was involved in producing TNF-α and IL-6;NF-κB signaling pathway inhibitor BAY11-7082 affected Brucella intracellular survival.So rough cattle Brucella strains RB51 intracellular survival and NF-κB signaling pathway activity were closely related.The results laid the foundation for the further study of Brucella intracellular pathogenesis,also provided scientific basis for the research of new drugs to Brucella,and prevention and treatment of brucellosis.     

1种布鲁氏菌微滴式数字PCR检测方法的建立

本研究旨在构建1种布鲁氏菌（Brucella）微滴式数字PCR方法（droplet digital PCR,ddPCR）。以布鲁氏菌BCSP31基因为靶基因,选取保守区域设计引物和探针,构建了布鲁氏菌微滴式数字PCR方法。然后优化了ddPCR反应中的引物、探针浓度及退火温度,并进行方法的灵敏度、特异性和重复性试验。结果表明,ddPCR的最佳引物和探针浓度分别为900 nmol·L^-1和250 nmol·L^-1,最佳的退火温度为58℃。ddPCR的线性良好,最低检测下限为1.12 copies·μL^-1,与大肠杆菌O：157菌株等其他4种细菌无交叉反应,而且批内重复性和批间重复性都较好。综上表明,本研究建立的ddPCR方法灵敏度高、特异性强,可对布鲁氏菌感染的临床样品进行定量检测。     

The Brucella genome at the beginning of the post-genomic era

The year 2002 began with the publication of the first complete genome sequence for a Brucella species, that of the two replicons of B. melitensis 16M. Hopefully in 2002, the complete genome of B. suis 1330, and, perhaps, a B. abortus strain will be published. This is the culmination of over 30 years investigation of the composition, structure, organisation and evolution of the Brucella genome. Brucella research must now adapt to the new challenges of the post-genomic era.     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

Construction of Brucella ClpS Gene Mutant Strain and Its Effect on Virulence

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 10^3.93 CFU/g spleen,which was significantly lower than that of 2308 strain (10^6.68 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.     

Brucella intracellular life: from invasion to intracellular replication

Brucella organisms are pathogens that ultimate goal is to propagate in their preferred niche, the cell. Upon cell contact the bacteria is internalized via receptor molecules by activating small GTPases of the Rho subfamily and by a moderate recruitment of actin filaments. Once inside cells, Brucella localizes in early phagosomes, where it avoids fusion with late endosomes and lysosomes. These early events require the control of Rab small GTPases, and cytokines such as the G-CSF. Then, the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum, where it extensively replicates. Some of the bacterial molecular determinants involved in the internalization and early events after ingestion are controlled by the BvrS/BvrR two component regulatory system, whereas the intracellular trafficking beyond this early compartments are controlled by the VirB type IV secretion system. Once inside the endoplasmic reticulum, Brucella extensively replicates without restricting basic cellular functions or inducing obvious damage to cells. The integrity of Brucella LPS on the bacterial surface is one of the required factors for Brucella intracellular survival, and therefore for virulence.     

布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响

【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...     

哈萨克绵羊DRB1基因外显子1和4多态性与布鲁氏菌病的相关性研究

试验旨在对哈萨克绵羊DRB1基因外显子1和4多态性与布鲁氏菌病的相关性进行研究。使用虎红平板凝集试验（RBPT）对试羊的血清进行血清学检测，参考GenBank中绵羊MHC ClassⅡ区DRB1基因序列（登录号：NC_040271.1），对其外显子1和4片段设计引物，采用PCR-SSCP和DNA测序技术对230只哈萨克绵羊的DRB1基因进行多态性检测，分析其多态位点与哈萨克绵羊布鲁氏菌易感性之间的关系。RBPT检测发现66只哈萨克绵羊为布鲁氏菌感染阳性，阳性检出率为28.7%。外显子1片段存在一个SNP位点（F1-G22A），测序确定两种基因型（GG、GA），优势等位基因和基因型分别为G、GG，F1-G22A多态位点的易感基因型为GA。卡方检验表明，哈萨克绵羊DRB1基因F1-G22A多态位点与布鲁氏菌易感性的相关性不显著（P>0.05）。通过生物信息学在线软件分析得出，F1-G22A多态位点导致了RNA二级结构的改变和最小自由能的降低，引起了蛋白质二级结构的改变。DRB1基因外显子4片段未发现SNPs。由此得出，哈萨克绵羊DRB1基因F1-G22A多态位点与布鲁氏菌易感性可能存在一定的相关性。     

The Correlation Between Polymorphisms of Exon1 and Exon4 of DRB1 Gene and Brucellosis in Kazakh Sheep

The purpose of this experiment was to study the correlation between exon 1 and 4 polymorphisms of DRB1 gene and brucellosis in Kazakh sheep.Using RBPT serological tests to try sheep serum,reference in GenBank sheep MHC Class Ⅱ area DRB1 gene sequences (Accession No.:NC_040271.1),the exon 1 and 4 pieces designed primers,using PCR-SSCP and DNA sequencing technology to 230 Kazak sheep DRB1 gene polymorphism detection,analyze its polymorphism loci and the relationship between the Kazak sheep Brucella susceptibility.The results showed that 66 Kazakh sheep were positive for Brucella in RBPT test,and the positive detection rate was 28.7%.There was one SNP locus (F1-G22A) in exon 1 fragment,and sequencing determined two genotypes (GG and GA),the dominant allele and genotype were G and GG respectively,and the susceptibility genotype of the polymorphisms of F1-G22A was GA.Chi-square test showed that there was no significant correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep (P>0.05).According to the analysis of bioinformatics online software,the F1-G22A polymorphic sites lead to the change of RNA secondary structure and the decrease of minimum free energy,and lead to the change of protein secondary structure.No SNPs were found in DRB1 exon 4 fragment.Therefore,there might be a certain correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep.     

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 μg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to β-lactam antibiotics. PBP2 complements the activity of PBP1. New β-lactam antibiotics and improved vaccines might be developed based on this property.     

牛种布鲁氏菌ClpS基因突变株的构建及其对毒力的影响

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10^3.93 CFU/g脾脏,显著低于亲本株2308（10^6.68 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。     

Discovery of Brucella virulence mechanisms using mutational analysis

This overview is written with the aim of providing an introduction to and historical perspective on naturally occurring and experimentally derived Brucella mutants. Spontaneous or naturally occurring mutants have proven of value over the last century in combating animal brucellosis. The most effective of these, S19, Rev-1 and RB51 have been used as vaccines in animals, but have drawbacks that have limited their effectiveness in some hosts including humans. The spontaneous appearance of these mutants has never been genetically characterized, but is now possible with the advent of genome sequencing. However, it is possible that these strains contain multiple genetic changes and identifying the relevant defects may be exceedingly difficult. Despite early sequencing initiatives Brucella virulence remains unresolved and a limited number of molecular systems have been identified that specifically enhance growth in host cells, the so-called virulence genes. Instead, the Brucella appear to have preserved a plethora of metabolic functions from their ancestors that enhance growth and survival in specific or varied environments, some of which are even duplicated. Direct and controlled mutagenesis of Brucella remains a valuable experimental approach to characterize the role of these genes in survival and virulence.     

布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

为探究Ⅳ型分泌系统在布鲁氏菌（Brucella）感染过程中的作用,深入了解布鲁氏菌Ⅳ型分泌系统在疫苗开发中的潜力,本研究以牛种布鲁氏菌A19疫苗株为研究对象,使用A19 VirB启动子缺失株感染小鼠树突状细胞（DCs）,通过菌落计数（CFU）评估Ⅳ型分泌系统对布鲁氏菌黏附侵袭及胞内生存的影响,同时对感染的细胞进行RNA和总蛋白的提取,分别通过实时荧光定量PCR和Western blotting检测自噬基因Beclin-1的转录和表达情况;收集感染后的细胞上清液,利用ELISA检测炎症因子白细胞介素-6（IL-6）和IL-10的分泌水平。黏附侵袭结果显示,布鲁氏菌VirB启动子缺失株与亲本株A19的黏附侵袭水平无显著差异（P>0.05）;胞内生存试验发现,感染的4 h,布鲁氏菌VirB启动子缺失株的胞内存活能力显著低于亲本株A19（P<0.05）,感染后0、24和48 h极显著低于亲本株A19（P<0.01）;实时荧光定量PCR和Western blotting结果显示,感染后4、8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生Beclin-1的水平极显著高于亲本株A19（P<0.01）;ELISA结果显示,感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生IL-6的水平显著高于亲本株A19（P<0.05）,而在感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞分泌IL-10的水平显著低于亲本株A19（P<0.05）,感染24 h时极显著低于亲本株A19（P<0.01）。综上所述,当VirB启动子缺失后,布鲁氏菌对DCs的黏附侵袭能力并未明显改变,但显著降低了布鲁氏菌在DCs内的存活能力,提升了DCs的自噬水平,促进了DCs IL-6的分泌,抑制了IL-10的分泌。本研究初步探究了布鲁氏菌Ⅳ型分泌系统在感染DCs过程中的生物学作用,为后续布鲁氏菌疫苗改造研究奠定了理论基础。     

线粒体融合蛋白2基因干扰与过表达对布鲁氏菌诱导巨噬细胞凋亡的影响

【目的】试验旨在探究线粒体融合蛋白2(MFN2)基因干扰与过表达对布鲁氏菌诱导小鼠巨噬细胞凋亡的影响。【方法】利用RNA干扰技术设计3条特异性针对MFN2基因的siRNA序列(siMFN2-450、siMFN2-1661、siMFN2-2275)和1条阴性干扰序列(siMFN2-Negative);利用空质粒pcDNA3.1-EGFP通过过表达技术构建1个重组质粒pcDNA3.1-EGFP-MFN2。双酶切法鉴定过表达重组质粒pcDNA3.1-EGFP-MFN2,通过实时荧光定量PCR和Western blotting检测筛选最佳干扰序列并鉴定重组质粒的过表达效率,使用筛选出的最佳干扰序列和鉴定过表达后的重组质粒分别构建MFN2基因干扰及过表达的转染巨噬细胞模型;用牛种布鲁氏菌疫苗株A19侵染巨噬细胞,按照细菌数∶细胞个数=100∶1的比例侵染24 h,通过实时荧光定量PCR和Western blotting检测B淋巴细胞瘤-2(Bcl-2)相关X蛋白(BAX)的表达情况,用流式细胞术检测细胞凋亡情况。【结果】双酶切结果显示,pcDNA3.1-EGFP-MFN2过表达重组质粒构建成功;...     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.