布鲁氏菌IV型分泌系统效应蛋白BPE043的功能和致病机制研究

目的 研究布鲁氏菌IV型分泌系统效应蛋白BPE043在布鲁氏菌感染和胞内生存过程中的作用。方法 以羊种布鲁氏菌104株为起始菌株,通过同源重组的方法构建布鲁氏菌BPE043基因缺失株。将布鲁氏菌野生株104和突变株104ΔBPE043在相同的起始浓度下培养,观察它们生长变化的趋势;用野生株和突变株分别感染小鼠,构建小鼠体内感染模型,测定小鼠脾重并计算脾脏细菌载量;利用免疫共沉淀技术筛选BPE043蛋白的宿主互作蛋白。结果 成功构建了布鲁氏菌BPE043基因缺失株。与野生株相比,突变株104ΔBPE043在体外培养的生长趋势和野生株基本相同;小鼠感染实验显示,在感染1周后,两组小鼠脾均肿大,但突变株104ΔBPE043感染组小鼠脾重低于野生株104感染组小鼠脾重;在感染后两周,两组小鼠脾肿大现象开始减轻。在细菌载量方面,在感染1周后,突变株104ΔBPE043感染小鼠的脾脏细菌载量低于野生株104感染小鼠;在感染后2周,突变株感染组的脾细菌载量下降趋势更明显。免疫共沉淀实验显示BPE043蛋白与鼠源小分子GTP结合蛋白Rab4和Rab11存在相互作用。结论 布鲁氏菌IV型分泌系统效应蛋白BPE043是布鲁氏菌重要的毒力因子。本研究为进一步探索BPE043基因在布鲁氏菌感染和胞内生存中的作用奠定了基础。     

恒河猴人工感染BRUCELLA OVIS后 病理学观察及病原回收

选用健康公恒河猴6只分3组每组2只,以Brucella ovis进行人工1亿、0.1亿、0.01亿菌量感染后35天再分别以10亿、5亿和1亿菌感染。结果在180天开始1、2组猴出现明显临床症状第3组则症状轻微。300天扑杀后可见附睾,睾丸、精索、淋巴结、脾、肝、肾、肺等器官和组织有典型病变;并在病变器官中分离到B.ovis培养物。     

布鲁氏菌S2株菌壳的制备研究

目的 利用温控表达调节系统在布鲁氏菌中诱导表达裂解基因E,制备布鲁氏菌S2株菌壳,监测其裂解效率,并观察菌壳形态。方法 从pBV220::E中PCR扩增温控裂解盒和裂解基因E融合片段,克隆至pBBR1MCS-2载体上,构建重组裂解质粒pBBR1MCS-E,并将其电转化至布鲁氏菌S2株,构建重组菌S2(pBBR1MCS-E)。通过28℃至42℃升温诱导培养制备布鲁氏菌菌壳,绘制生长曲线和裂解曲线,计算裂解率,并电镜观察菌壳形态。结果 成功构建了重组裂解质粒pBBR1MCS-E并制备了布鲁氏菌菌壳。绘制出了布鲁氏菌裂解曲线,布鲁氏菌菌壳的裂解率达99.99%,经高渗溶液冻融后可完全消除其中残存的活菌。电镜观察表明布鲁氏菌菌壳保持有细胞的基本形态,但由于细胞内容物外流而使细胞发生变形和皱缩。结论 本研究成功制备出了布鲁氏菌菌壳,为进一步研究布鲁氏菌菌壳的特性奠定了基础。     

短小棒状杆菌对布鲁氏菌组分苗免疫影响的初步试验观察

本文报告了短小棒状杆菌(Corynebacterium parvum)对布鲁氏菌组分苗免疫影响的初步试验研究,在本次试验中,布鲁氏菌组分苗A、E组分和短小棒状杆菌苗联合注射时未引起明显的非特异性免疫增强作用。组分苗A保护力为75％,A组分与棒状杆菌联合注射的保护力为88.9％;组分苗E保护力为90％,E组分与棒状杆菌苗联合注射保护力为100％,它们之间无显著性差异。     

布鲁菌液体气溶胶肺部感染途径小鼠模型建立及毒理评价

目的建立布鲁菌液体气溶胶感染小鼠模型并进行毒理评价。方法选取6~8周龄BALB/c雌鼠,经肺递送和滴鼻2种途径感染布鲁菌S19-CN,分别于第0、2、4、6、8周,观察并记录临床体征,采集样本,分别对脾脏和肺脏载菌量计数及抗体(血清)和细胞因子(血清和肺匀浆)进行检测。结果与空白对照组比较,感染后,肺递送组、滴鼻组小鼠均未见病症;体质量无显著下降;脾重分别在感染后第2周、第4周达到峰值;肺载菌量均在第2周达到峰值,约107 CFU/g;血清抗体均在感染后8周内维持较高水平,肺递送组和滴鼻组的血清和肺匀浆中均可检测到高浓度细胞因子IFN-γ、TNF-α,且肺递送组峰值高于滴鼻组,与空白对照比较差异有统计学意义(P均0.05)。结论本研究通过布鲁菌S19-CN液体气溶胶肺递送感染BALB/c小鼠,成功建立布鲁菌液体气溶胶感染小鼠模型。S19-CN引起BALB/c小鼠慢性感染,脾肿大明显,毒理作用显著;同时激发了较强的细胞免疫和体液免疫反应。     

马鹿布氏杆菌25kDa外膜蛋白基因的克隆、序列分析及其表达研究

目的克隆马鹿布氏杆菌ML分离株25kDa外膜蛋白(Omp25)基因,并在大肠杆菌中表达。方法运用RT-PCR技术从马鹿布氏杆菌分离株中扩增出Omp25基因,将其克隆入pMD18-T中进行核苷酸序列测定和遗传变异分析,并将目的基因亚克隆到大肠杆菌表达载体pET28a中诱导表达。结果该基因全长642bp,编码213个氨基酸;其中前23个氨基酸残基构成信号肽。在推导的氨基酸序列中,存在两个跨膜区,但没有潜在的N-联糖基化位点。与GenBank中已经登录的布氏杆菌其他11个分离株相比,马鹿布氏杆菌分离株Omp25基因变异较小,以散在的点突变为主,Omp25基因核苷酸和推导氨基酸序列的同源性分别为99.1%-99.4%和99.2%-99.5%之间。转化重组质粒pETOMP25的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出具有反应原性的目的蛋白,表达量占菌体蛋白的18.6%。结论马鹿布氏杆菌分离株与流产型布氏杆菌其它分离株Omp25基因同源性很高,可能是一个毒力较强的野毒株。     

Phenol-insoluble vaccine against human brucellosis: evaluation of the immunogenic power in guinea pigs]

A phenol insoluble extract vaccine proposed to immunize men against brucellosis was tested for its ability in protecting guinea pigs against challenge with virulent Brucella abortus strain 2308. Living attenuated Brucella abortus strain 19 and B. melitensis Rev. 1 were included in the experiment for comparison. Ninety three animals were vaccinated in each group and subdivided in subgroups of 31 for challenge with 10(4), 10(3) and 10(2) colony forming units of virulent B. abortus 2308. A global analysis of the results showed protection of 11.9%, 65% and 95% in animals vaccinated with phenol insoluble extract, strain 19 and Rev. 1, respectively.     

Improved Immunogenicity of Tetanus Toxoid by Brucella abortus S19 LPS Adjuvant

Background: Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. Objectives: To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. Methods: LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for the conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund’s adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Results: Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. Conclusion: These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.     

Evaluation of a neuraminidase-specific influenza A virus vaccine in children: antibody responses and effects on two successive outbreaks of natural infection.

Three groups of children were immunized with an inactivated Port Chalmers (H3ChN2Ch) influenza vaccine (group A), a neuraminidase-specific (Heq1N2Ch) influenza vaccine (group B), or a placebo. Immunization induced seroconversion for H3Ch and N2Ch-specific antibody in group A and for N2Ch antibody in group B. The protective efficacies observed against naturally acquired illness with the Port Chalmers strain of influneza A virus were 68.7% and 37.4% in groups A and B, respectively, in comparison to the placebo group, and those against illness produced by the subsequent outbreak of the Victoria strain were 80.0% and 72.7%. These data support the role of neuraminidase-specific immunization in protection against influenza. Although the degree of protection after vaccination with the Heq1N2Ch vaccine was less than that provided by the biphasic H3ChN2Ch vaccine against the Port Chalmers strain, it appeared to be similar in the two vaccine study groups against the Victoria strain.     

牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立

目的 建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方-法。方法 分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行 Realtime-PCR扩增,评价该方-法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方-法的敏感性。结果 本方-法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10² copies/μL和10³ copies/μL。该方-法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论 本研究建立的布鲁氏菌双重Realtime-PCR方-法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。