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猪链球菌-副猪嗜血杆菌二联亚单位疫苗在小鼠的免疫效力分析 总被引：1，自引：0，他引：1
本研究旨在开发一种用于防制猪链球菌病和副猪嗜血杆菌病的新型二联基因工程疫苗,并对其免疫效应进行评价.选择猪链球菌2型蛋白溶血素SLY、HP0197和副猪嗜血杆菌外膜蛋白D15和HPS06257 4种蛋白,重组表达并纯化,添加一定比例的佐剂制成含有不同组分的亚单位疫苗.研究表明该二联亚单位疫苗及各单苗能够引起小鼠良好的体液免疫,并能保护机体分别抵抗致死剂量猪链球菌2型(SS2)和副猪嗜血杆菌(HPS)强毒株的攻击.此外,调理吞噬试验表明疫苗高免血清能够有效杀死细菌,并赋予机体被动抵抗SS2和HPS感染的能力.研究证实制备的猪链球菌-副猪嗜血杆菌二联亚单位疫苗在小鼠具备良好的免疫保护效力. 相似文献
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袁硕峰 《畜牧兽医科技信息》2010,(6):13-14
猪链球菌是一种重要的人畜共患病原体。毒力因子以及抗原性蛋白的发掘与探索有助于深入研究猪链球菌病的致病机制,并为疫苗研究提供候选基因序列和蛋白片段。国内外学者建立了多种发掘猪链球菌新的毒力因子和抗原性蛋白的方法:转座子诱变缺陷株筛选法、免疫蛋白质组学技术、等位基因置换法、体内互补法、构建和筛选了基因文库、抑制性差减杂交试验等。 相似文献
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本试验通过分子克隆技术分别构建了单独表达猪繁殖与呼吸综合征病毒（PRRSV）GP5基因以及PRRSVGP5基因和猪IL-18基因共表达的重组核酸疫苗质粒（pEGFP—GP5和pEGFP—ILl8-GP5）,并进行仔猪免疫原性研究,对构建的PRRSV核酸疫苗所诱导的体液免疫和细胞免疫水平进行检测,进一步研究了PRRSV核酸疫苗免疫效果以及猪IL-18基因对PRRSV核酸疫苗免疫调节作用的影响。同时,调查了商业上应用不同类型的PRRSV疫苗诱导的免疫效果,并与核酸疫苗免疫效果进行比较。结果表明,IL-18作为免疫佐剂在疫苗免疫猪后诱导的病毒特异性细胞免疫反应方面具有很好的调节作用,共表达IL18-GP5蛋白能够明显的改善DNA疫苗的免疫效力,增强抗PRRSV的免疫保护。因此,DNA疫苗做为一种新一代疫苗可用于对抗高致病性PRRSV感染。 相似文献
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猪链球菌病流行病学及其疫苗研究现状 总被引：8，自引：1，他引：7
猪链球菌病流行无明显的季节性,一年四季均可发生,尤其是重症猪链球菌2型感染暴发时,致病性强,传播迅速,猪病死率高。该病同时可通过破损皮肤如伤口或擦伤传染给人,也可通过呼吸道感染人,严重感染时可引起人的死亡。控制猪链球菌病的感染,重在预防。不同类型的疫苗已研制成功或正在开发。近年来,基因工程疫苗如重组亚单位疫苗,细菌载体疫苗等新型疫苗的研究具有广泛应用前景。虽然猪链球菌病在猪群中的流行早有报道,但人类感染的报道较少,认识较局限。文章主要对该病的病原特性。流行病学及疫苗的研究做了系统的阐述,为该病的研究提供参考。 相似文献
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Streptococcus equi ssp. zooepidemicus (SEZ) is an important swine pathogen and responsible for a wide variety of infections in many animal species. FabF was a novel protein identified in the previous study. However, its protective efficacy remained to be evaluated. In this study, recombinant fabF of SEZ was expressed and showed a strong immunoreactivity with mini-pig convalescent sera. Study in mice revealed that the recombinant protein induced a marked antibody response and protected 80% of mice against SEZ infection. The hyperimmune sera against fabF could efficiently kill the bacteria in the phagocytosis test. In addition, it was also found that anti- fabF antibodies can significantly inhibit the formation of SEZ biofilm. These study suggest that fabF may represent immunogens of interest for vaccine development against SEZ infection. 相似文献
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OBJECTIVE: To determine whether previously unidentified variations of the SzP protein of Streptococcus equi subsp zooepidemicus were present in horses with various clinical signs of infection and whether any relationship could be identified between SzP protein variants and naturally occurring clinical conditions. SAMPLE POPULATION: 23 isolates of S equi subsp zooepidemicus were recovered from specimens of horses with various clinical conditions and used as a representative population of isolates for evaluation of different SzP protein variants. PROCEDURE: Genetic heterogeneity of the isolates was demonstrated by repetitive extragenic palindromic-polymerase chain reaction analysis. The SzP gene was sequenced and the presumed protein sequence determined for each isolate. Characteristics of the SzP proteins were compared among the isolates and in relation to the clinical conditions of horses from which they were recovered. RESULTS: The signal peptide types, number of proline-glutamic acid-proline-lysine repeats, and anchor sequences were consistent with those previously described for the SzP protein. Many of the isolates clustered with 5 previously described types on the basis of the hypervariable region of the SzP protein. One additional variant, which represented 8 of the isolates, was identified. Particular motifs in the hypervariable region accounted for many of the differences among hypervariable types. CONCLUSIONS AND CLINICAL RELEVANCE: The SzP protein appears to be limited to a selected number of types. Variations in the SzP protein are frequently determined on the basis of different motifs rather than random amino acid substitutions. There does not appear to be any association of SzP protein variations and clinical manifestations of infection in horses. 相似文献
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马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响 总被引：1，自引：1，他引：0
旨在研究马链球菌兽疫亚种（Streptococcus equi ssp.zooepidemicus，SEZ）烯醇化酶（enolase，Eno）对小鼠肺泡巨噬细胞（RAW264.7）吞噬能力的影响。通过构建原核表达质粒获得重组烯醇化酶（rEno），采用台盼蓝活细胞计数法，判定在不同处理浓度和时间下rEno蛋白对RAW264.7细胞的细胞毒性。将rEno蛋白与RAW264.7细胞共孵育后，用SEZ作用于细胞并检测细胞吞菌数量，判断RAW264.7细胞对SEZ的吞噬活性。进一步通过活细胞稳定同位素标记技术（SILAC）和蛋白质谱分析技术（LC-MS/MS），筛选到RAW264.7细胞中可能与SEZ Eno存在相互作用的候选蛋白。结果发现，10 μg·mL-1 rEno蛋白处理对RAW264.7细胞有明显的细胞毒性，且10 μg·mL-1 rEno蛋白处理RAW264.7细胞2和4 h可显著抑制其对SEZ的吞噬作用（P<0.01、P<0.05）。初步筛选到RAW264.7细胞中动力蛋白激活蛋白亚单位蛋白（dynactin subunit protein 2，Dctn）、整合素α-M蛋白（integrin alpha-M）等17种可能与Eno发生互作的蛋白。本研究获得了rEno重组表达蛋白，发现rEno可减少RAW264.7细胞对SEZ的吞噬，互作蛋白的初步筛选也为进一步揭示Eno在SEZ抗吞噬中的作用机制奠定了基础。 相似文献
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CHEN Kaiwen HUA Chengwei YUAN Chen PAN Fei LIN Huixing FAN Hongjie MA Zhe 《畜牧兽医学报》1956,51(10):2576-2583
To investigate the effect of enolase (Eno) of Streptococcus equi ssp. zooepidemicus (SEZ) on phagocytosis of mouse alveolar macrophages (RAW264.7). Recombinant enolase (rEno) was obtained by constructing prokaryotic expression plasmid, and the cytotoxicity of rEno protein on RAW264.7 cell proliferation was determined by trypan-blue living cell count method. After the rEno protein was incubated with RAW264.7 cells, SEZ was applied to the cells and the quantity of bacteria being phagocytosed was detected to determine the phagocytic activity of RAW264.7 cells. Further, candidate proteins that might interact with SEZ Eno in RAW264.7 cells were screened by live cell stable isotope labeling (SILAC) and protein spectrum analysis (LC-MS/MS). It was found that protein treatment (rEno,10 μg·mL-1) had significant cytotoxic effects on RAW264.7 cells. Treatment of RAW264.7 cells with 10.0 μg·mL-1 rEno protein for 2 and 4 hours could significantly inhibit the phagocytosis of RAW264.7 cells (P<0.01, P<0.05). In RAW264.7 cells, dynactin subunit protein 2 (Dctn), integrin alpha-M and about 17 proteins that might interact with Eno were preliminarily identified as rEno interaction proteins. The rEno recombinant expression protein was obtained in this study, and it could reduce the phagocytosis of RAW264.7 cells to SEZ. Preliminary screening of interacting proteins also laid a foundation for further revealing the mechanism of Eno in the anti-phagocytosis of SEZ. 相似文献
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Anzai T Walker JA Blair MB Chambers TM Timoney JF 《American journal of veterinary research》2000,61(2):162-166
OBJECTIVE: To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. SAMPLE POPULATION: 5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. PROCEDURE: Variable M-like protectively immunogenic SzP proteins of 5 isolates of S. zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 healthy horse and 9 horses and donkeys with pneumonia were sequenced and compared. Cell-associated hyaluronic acid concentration and resistance to phagocytosis of some isolates were measured. RESULTS: Tonsils of each healthy horse were colonized by several SzP phenotypes similar to those of foals or donkeys with pneumonia. In contrast, multiple isolates from animals with pneumonia had the same SzP phenotype, indicating infection by a single strain or clone. Analysis of the SzP sequence confirmed that differences in immunoblot phenotype were associated with sequence differences and that several SzP genotypes were in healthy horses and animals with pneumonia. Isolates with high concentrations of cell-associated hyaluronic acid were more resistant to phagocytosis. CONCLUSIONS AND CLINICAL RELEVANCE: An SzP-specific immunoblot is a useful, sensitive measure of diversity among strains of S. zooepidemicus. Single strains with SzP phenotypes similar to those found in tonsils of healthy horses cause pneumonia. Because of the diversity of SzP phenotype and genotype among isolates from animals with pneumonia, SzP phenotype is not an important determinant of invasiveness or epizootic capabilities. 相似文献
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Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is responsible for a wide variety of infections in many species. Attempts to control the infection caused by this agent are hampered by a lack of effective vaccines and useful diagnostic kits. Surface proteins of bacterial species are usually involved in interaction with host and hopefully act as biomarkers for serodiagnosis and subunit vaccine components. In this study, the surface proteins of SEZ C55138 strain were systematically identified by surface shaving with trypsin and a total of 20 surface associated proteins were found. Further analysis of five selected novel proteins (SzM, FBP, SAP, CSP and 5'-Nu) revealed that they all expressed in vivo and their recombinant derived proteins could be reactive with convalescent sera. These identified immunogenic surface proteins have potential as SEZ vaccine candidates and diagnostic markers. 相似文献
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根据已发表的马链球菌兽疫亚种MGCS10565酮基转移酶(transketolase)的基因序列,设计并合成引物。以ATCC35246株基因组DNA为模板,通过PCR技术,扩增出目的基因并定向克隆至表达载体pET-28a(+)中,然后将重组质粒转化入大肠杆菌BL21(DE3)中,分析并纯化表达产物。选用ICR小鼠作为实验动物模型,以纯化的重组融合蛋白通过皮下注射途径免疫小鼠,并用间接ELISA法监测小鼠血清中的抗体效价。结果表明重组蛋白免疫小鼠后能产生有效的免疫应答,血清中抗体水平有明显的升高。加强免疫2周后,以5LD50的ATCC35246强毒株攻击免疫组及对照组,结果免疫组小鼠的保护率可达37.5%。表明原核表达产物免疫ICR小鼠,可使其对同源菌株攻击产生一定的保护作用,在亚单位疫苗研制中具有潜在的应用价值。 相似文献
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Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy. 相似文献
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Anzai T Timoney JE Kuwamoto Y Wada R Oikawa M Higuchi T 《American journal of veterinary research》2002,63(9):1298-1301
OBJECTIVE: To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds. SAMPLE POPULATION: 10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia. PROCEDURE: Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, genotypes were obtained with primer sets from the SzP gene, followed by restriction endonuclease digestion of the amplicons. RESULTS: Unique genotypic patterns were obtained with a primer set designed from both ends of the structural gene and the restriction endonuclease DdeI. Forty-five isolates from the lymphoid tissue within the pharyngeal recess (ie, pharyngeal tonsil) of yearlings included 10 SzP genotypes and SzP phenotypes. Isolates from the trachea of each yearling were of a single genotype that was also present among isolates from the pharyngeal tonsil of the same horses. Isolates from tracheal aspirates of foals belonged to 14 genotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of the SzP gene by use of PCR-RFLP was effective for molecular typing of strains of S zooepidemicus in the study of respiratory tract disease in horses. Results of PCR-RFLP analysis indicate that a single strain of S zooepidemicus can migrate from the pharyngeal tonsil to the trachea at a high rate in horses undergoing long distance transportation. 相似文献
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Hebben M Duquesne V Cronier J Rossi B Aubert A 《Journal of Feline Medicine and Surgery》2004,6(2):111-118
Feline infectious peritonitis virus (FIPV) is a coronavirus that induces a fatal systemic disease mediated by an inappropriate immune response. Most previous vaccination attempts against FIPV were unsuccessful because IgG antibodies against the surface protein enhance the infection. However, two studies have shown that poxvirus vectors (vaccinia WR and canarypox) expressing only the FIPV membrane (M) protein can elicit a partially protective immunity which is supposed to be cell-mediated (Virology 181 (1991) 327; International patent WO 97/20054 (1997)). In our study, we report the construction of another poxvirus, the modified vaccinia virus Ankara (MVA), as an expression vector for the FIPV M protein. In this vector, the M gene has been inserted downstream a strong early/late promoter, whereas the two previously described poxviruses expressed the M protein during their early stage only. The immunogenicity of the recombinant MVA-M was evaluated in the murine model which revealed an effect of the vector on the Th1/Th2 balance. The vaccine was then tested in cats to evaluate its efficacy in an FIPV 79-1146 challenge. Vaccinated kittens developed FIPV-specific antibodies after immunization, however, none of them was protected against FIPV. Our results suggest a crucial role for the type of poxviral promoter that must be used to induce an effective immune response against FIPV. 相似文献