布鲁氏菌外膜蛋白OMP15.6表达及免疫反应原性测定

表达羊布鲁氏菌16M预测外膜蛋白OMP15.6,探索其作为诊断抗原的可能性。采用降落PCR方法,从羊布鲁氏菌16M基因组DNA中扩增出423bp的基因片段,将该片段克隆于原核表达载体PGEX-4T-2,构建重组表达载体。经IPTG诱导,SDS-PAGE检测,结果表明,在大肠杆菌中成功表达了外膜蛋白OMP15.6。经West-ern-blotting检测,该蛋白能与豚鼠抗布鲁氏菌阳性血清发生特异性免疫反应,为其之后的抗原性以及免疫原性研究奠定了良好的基础。     

布鲁氏菌脂多糖抗原纯化效果的比较

在纯度、产率、活性等方面比较了硫酸铵沉淀法、冷甲醇二次沉淀法、酶消化处理法纯化脂多糖(LPS)的效果.考马斯亮蓝染色和琼脂糖凝胶电泳定性分析结果证明酶消化处理和冷甲醇二次沉淀纯化的LPS纯度相近;酶消化处理LPS的产率较高;iELISA活性检测结果比较显示酶消化处理纯化的LPS活性较高,且纯化的全过程活性没有下降.重复性试验结果显示酶消化处理提纯方法的可重复性良好.     

布鲁氏菌外膜蛋白16基因的克隆及原核表达

为了成功克隆外膜蛋白16(outer membrane proteins 16, Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507 bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。     

布鲁氏菌外膜蛋白16单克隆抗体的制备及初步应用

旨在制备布鲁氏菌外膜蛋白16(OMP16)单克隆抗体,建立OMP16抗体竞争ELISA方法,为布病临床防控提供免疫血清学检测手段。本研究选取保守性高、免疫原性良好的布鲁氏菌OMP16,经原核表达获得携带GST或His标签的重组OMP16(rOMP16)。利用杂交瘤技术筛选可稳定分泌OMP16特异性单克隆抗体的杂交瘤细胞株,经亚型鉴定、细胞核型分析和抗体交叉反应性试验分析单抗性质。制备小鼠腹水并纯化,方阵滴定法建立布鲁氏菌OMP16抗体竞争ELISA方法。结果表明,成功构建OMP16原核表达系统,经表达、纯化获得rGST-uOMP16和rHis-OMP16。筛选到1株遗传稳定的OMP16特异性杂交瘤细胞株,命名为B7;经鉴定,该抗体亚型为IgM-κ型,可识别目的蛋白,与标签蛋白无交叉反应。建立了布鲁氏菌OMP16抗体竞争ELISA方法,该方法与试管凝集试验检测结果相比总体符合率为90.53%,显示出良好的一致性。本研究有望为布鲁氏菌病的临床防控提供特异性高、敏感性好的免疫血清学检测方法。     

牛布鲁氏菌OMP25的原核表达及表达产物的免疫学特性

目的研究布鲁氏菌外膜蛋白OMP25的克隆、测序和表达,并对其进行免疫特性检测。方法采用PCR技术对牛布鲁氏菌外膜蛋白OMP25基因进行了扩增和克隆测序,并成功构建了原核表达质粒pGEX-OMP25。将其转入E.coliBL21,经IPTG诱导表达,用电洗脱纯化后获得了高纯度的目的蛋白。纯化后的蛋白与弗氏佐剂乳化后免疫家兔得到了较高效价的抗血清。结论通过ELISA、Western-blotting分析及特异性检测试验,证明目的蛋白不仅具有抗原性且有良好的免疫反性。     

布鲁菌病研究进展

布鲁菌病是目前世界上流行最广、危害最大的人兽共患病之一,在全世界170多个国家和地区有人、畜布鲁菌病存在和流行.我国25个省(市、区)有人、畜布鲁菌病存在和流行.随着畜牧业的快速发展,布鲁菌病在多数疫区明显有回升趋势.2005年全国新发病19 664人,发病率为1.504/10万,超过历史最高记录.布鲁菌是细胞内寄生的革兰氏阴性菌,主要引起人的波状热和慢性感染以及动物睾丸炎和流产等,直接危害公共安全,造成严重的经济损失.近年来从多种海洋哺乳动物(海豹、海豚、鲸等)中也分离出布鲁菌.通过DNA同源性比较,发现海洋与陆地哺乳动物布鲁菌DNA的同源性高达90%以上.随着分子生物学的发展,对布鲁菌分子结构的研究不断获得新的发现,为进一步认识和控制布鲁菌病提供了可能.文章从布鲁菌病流行的历史与现状、布鲁菌的抗原分子生物特性、布鲁菌病的检测方法和布鲁菌病疫苗的现状等方面对布鲁菌病的研究进展做了介绍.     

布鲁氏菌抗体检测试纸条的研制和初步比较

本试验利用胶体金免疫层析技术原理,研制了布鲁氏菌抗体检测试纸条,并以布鲁氏菌阳性血清国家标准品进行了敏感性试验,确定了最低检出量,同时与有可能存在交叉反应的血清和阴性血清进行了特异性试验;然后将保存不同时间的试纸条在进行了敏感性和特异性试验,证明其稳定期可以达到15个月。选择临床牛羊血清30份,利用该试纸条与布鲁氏菌病虎红平板试验抗原进行同步检测,发现两种试剂的检测符合率为98%。试验证明,所研制的布鲁氏菌抗体检测试纸条敏感性高、特异性强、稳定期长。     

布鲁氏菌抗感染免疫研究进展

布鲁氏菌病(Brucellosis)是目前世界上流行最广,危害最大的人畜共患病之一,其致病菌为革兰氏阴性杆菌-布鲁氏菌,布鲁氏菌感染机体后能够刺激免疫系统,激发保护性免疫反应。在宿主对布鲁氏菌的抗感染免疫过程中,天然免疫和获得性免疫起着重要作用,其中天然免疫反应包括补体,噬中性粒细胞,NK细胞,巨噬细胞以及Nramp1的作用,而获得性免疫则包括CD4+,CD8+和γβT细胞分泌的IFN-γ等细胞因子和细胞毒性作用。由于布鲁氏菌为胞内寄生的革兰氏阴性杆菌,所以机体对抗其致病性的主要免疫方式为细胞免疫。     

精子表面抗原研究进展

从生殖细胞—精原细胞—精母细胞—精子细胞—精子的逐步发育过程中,细胞表面都携带抗原,并且随发育阶段与发育程度的不同其抗原数量、种类和分布发生相应改变。精子离开支持细胞进入精细管—睾丸网—睾丸输出管—附睾—附睾输出管直至射出体外,其表面抗原发生修饰(modifi cation)、被覆(coating)、插入(insertion)和丢失(loss)等方面的变化。因此研究精子表面抗原可以判断精子发育处于何种阶段和何种组织器官。应用单抗技术、免疫沉淀技术和免疫荧光技术,目前在精子表面已发现十几种抗原,大致分成白身抗原和非自身抗原两类。自身抗原是精子自身特有的,其它体细胞没有,具有组织特异性的抗原,由于血睾屏障的保护,不发生自身免疫反应。例如,睾丸炎的病人,血睾屏障被破坏,精子渗漏,引起外周血中抗精子抗体滴度升高,杀伤精子而导致雄性不育,这提示自身抗原可被人类用于制备避孕疫苗。非自身抗原来源于睾丸     

Risk factors for Brucella spp. infection In dairy cattle farms in Asmara, State of Eritrea

A cross-sectional study was conducted to identify risk factors for herd infection by Brucella spp. in dairy cattle in the suburbs of Asmara, Eritrea. Data were collected from 64 herds, randomly selected from a total of 99 herds with a minimum herd size of 9 cows. A questionnaire was used to gather data on management, hygiene and herd structure. Serum samples collected from all pregnant heifers, cows and bulls, were screened for Brucella infection by the Rose Bengal test (RBT), and all RBT-positive sera re-tested with the complement-fixation test (CFT) for confirmation. A seropositive herd was defined as one in which at least one animal tested positive in the CFT. There were 23 (36%) positive herds among the 64 studied. Both multiple logistic and multiple betabinomial regression modeling were used to analyze the data. Mixed-breed herds, compared to single (exotic)-breed herds, were found to be independently associated with increased herd seroprevalence (OR=5.2, 95% confidence interval 1.4–18.7) in the multiple logistic model with the herd infection status as the dependent variable. The importance of this variable was supported by the multiple betabinomial regression model (OR=3.3, 1.4–7.6) with animal-level prevalence within herd as the outcome variable. Both models also revealed the presence of a negative association between seropositivity and cattle stocking density.     

布鲁菌外膜蛋白及毒力因子研究进展

布鲁菌细胞膜的基本结构包括脂多糖和外膜蛋白,与细菌的毒力及免疫原性相关。文章描述了布鲁菌外膜蛋白分子结构的最新进展。该菌的外膜蛋白由第一组、第二组和第三组外膜蛋白构成。第一组外膜蛋白对维持布鲁菌外膜蛋白的结构起重要作用;第二组包括36 ku~38 ku外膜蛋白,为膜孔蛋白,由Omp2a和Omp2b基因编码,其中38 ku蛋白基因可能是一个与毒力相关的基因;第三组外膜蛋白包括31 ku和25 ku两个相关的蛋白,具有重要的免疫功能。31 ku蛋白属膜孔蛋白,25 ku蛋白还与毒力有关。文章也介绍了布鲁菌毒力因子研究的最新进展。     

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.     

布氏杆菌微滴数字PCR方法的建立

为建立一种布氏杆菌微滴数字PCR qPCR方法,对布氏杆菌病的定量诊断提供技术支持,在实时荧光PCR (qPCR)检测方法(T/CVMA 20-2020)的基础上,建立了布氏杆菌微滴数字PCR方法,并对方法的反应条件进行了优化,同时对其敏感性、特异性、重复性进行了评估.结果 显示:本方法的最低检测下限为2.6 copi...     

An aerosolized Brucella spp. challenge model for laboratory animals

To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M) in a laboratory animal model of brucellosis, dosages of 10(3)-10(10) colony forming units (CFU) were nebulized to mice. Although tissue weights were minimally influenced, total CFU per tissues increased beginning at 10(6)-10(7) CFU dosages, with 10(9) CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery. At 12 weeks after vaccination with 10(7) CFU of B. abortus strain RB51 (SRB51) or saline (control), mice were challenged intraperitoneally (i.p.) (6.4 x 10(4) CFU) or via aerosol (1.76 x 10(9) CFU) with S2308. Mice vaccinated with SRB51 had reduced (P < 0.05) splenic, liver and lung colonization (total CFU and CFU/g) after i.p. challenge with S2308 as compared with control mice after i.p. S2308 challenge. Control and SRB51-vaccinated mice did not differ (P > 0.05) in splenic, liver or lung colonization after aerosol S2308 challenge. Failure to demonstrate vaccine protection was not because of a high aerosol challenge dosage as colonization of spleen and liver tissues was lower (P < 0.05) after aerosol challenge when compared with control mice after i.p. S2308 challenge.     

Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas,USA

Various tissues, nasal swabs, urine and blood samples were collected from 376 feral swine at two federally inspected abattoirs in Texas during six separate sampling periods in 2015. Samples were tested for Brucella spp. by culture and serology. Brucella spp. were cultured from 13.0% of feral swine, and antibodies were detected in 9.8%. Only 32.7% of culture‐positive feral swine were also antibody positive, and 43.2% of antibody‐positive feral swine were culture positive. Approximately, the same number of males (14.0%) and females (12.1%) were culture positive, and slightly more males (10.5%) than females (8.7%) were antibody positive. Our results indicate that serology likely underestimates the prevalence of feral swine infected, and that those who come in contact with feral swine should be aware of the symptoms of infection with Brucella spp. to ensure prompt treatment.     

布鲁氏菌A、M因子血清的研制

研制牛种布鲁氏菌A因子血清和羊种布鲁氏菌M因子血清,用于光滑型布鲁氏菌特异性种属鉴定。将灭活牛种布鲁氏菌2308株及羊种布鲁氏菌16M株免疫1.5~2 kg家兔各5只制备高免血清。对高免血清采用抗原交叉吸附的方法,制得A、M因子血清,分装冻干后置-20℃保存。微量试管凝集试验结果显示:研制的A因子血清对16M抗原在1∶2.5时无凝集现象,对2308抗原凝集价为1∶320;研制的M因子血清对2308抗原在1∶2.5时无凝集现象,对16M抗原凝集价为1∶160。玻片凝集试验结果显示:研制的A、M因子血清对异种抗原无凝集现象。试验表明,研制的A、M因子血清具有特异性强、效价高的特点,可辅助光滑型布鲁氏菌特异性种属鉴定。     

雄性特异性抗原的研究进展

雄性特异性抗原为雄性动物特有,是引起同种雌性动物免疫排斥反应的物质的总称。这类物质广泛存在于哺乳动物及其他脊椎动物。依据其检测方式的不同,可分为细胞毒性T细胞相关性雄性特异性抗原和血清学相关性雄性特异性抗原。目前,已初步确定了几个细胞毒性T细胞相关性雄性特异性抗原的候选基因及抗原肽序列,它们都位于Y染色体上。对于血清学相关性雄性特异性抗原的研究则还处于初级阶段。近年发现了细胞毒性T细胞相关性雄性特异性抗原的抗体,揭示此两类物质具有内在的联系。     

Regulation of Brucella virulence by the two-component system BvrR/BvrS

The Brucella BvrR/BvrS two-component regulatory system is highly similar to the regulatory and sensory proteins of Sinorhizobium and Agrobacterium necessary for endosymbiosis and pathogenicity in plants, and very similar to a putative system present in the animal pathogen Bartonella. Mutations in the bvrR or bvrS genes hamper the penetration of B. abortus in non-phagocytic cells and impairs intracellular trafficking and virulence. In contrast to virulent Brucella, BvrR/BvrS mutants do not recruit small GTPases of the Rho subfamily required for actin polymerization and penetration to cells. Dysfunction of the BvrR/BvrS system alters the outer membrane permeability, the expression of several group 3 outer membrane proteins and the pattern of lipid A acylation. Constructs of virulent B. abortus chimeras containing heterologous LPS from the bvrS⁻ mutant demonstrated an altered permeability to cationic peptides similar to that of the BvrR/BvrS mutants. We hypothesize that the Brucella BvrR/BvrS is a system devoted to the homeostasis of the outer membrane and, therefore in the interface for cell invasion and mounting the required structures for intracellular parasitism.     

The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome

The successful co-existence of each Brucella spp. with its preferred host is the outcome of ancient co-evolutionary relationships and selection pressures that often result in a stalemate where the pathogen has evolved to survive within the biological systems of the host, and the host has evolved innate and acquired immune systems which allow controlled survival of infection by the pathogen, ultimately supporting the survival of the host-pathogen system. In general, Brucella spp. have evolved a similar fundamental pathogenesis of facultative intracellular parasitism though the predominant route of natural exposure varies from oropharynx to genital tract, as does the preferred tissue and cellular tropism, e.g. non-professional placental trophoblasts, fetal lung, professional macrophages of reticulendothelial system, and the male and female reproductive tracts. The morphogenesis of the pyogranulomatous lesions stimulated by Brucella reflects the nature of the persistent parasitism, i.e. genome versus genome. The question is, how can this perplexing array of survival mechanisms be unraveled? Fortunately, the integration of real-time image analysis, cell biology, genome-wide analysis, proteomics and bioinformatics holds the most promise ever for the global analysis of the Brucella infectious process and the host:pathogen interface leading to a clearer understanding of the interactions of these biological systems. These discoveries will be expected to provide a frameshift in rationales for interrupting and/or controlling brucellosis at host and/or pathogen levels.