百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌     

家兔支气管败血波氏杆菌重组百日咳杆菌粘附素（PRN）蛋白的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）PRN蛋白,并以此建立检测Bb抗体的间接ELISA方法。方法参照GenBank公布的猪源支气管败血波氏杆菌prn基因序列（AY376325）设计了一对特异性引物,PCR扩增出相应的核苷酸片段。将PCR扩增产物连接至原核表达载体pGEX-4T-1中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白PRN作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数等,建立检测支气管败血波氏杆菌抗体的间接ELISA方法。结果成功克隆了prn全基因序列,并在E.coliBL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白PRN具有良好的抗原性。应用重组蛋白PRN为抗原建立了检测Bb血清抗体的间接ELISA诊断方法。试验确定重组蛋白PRN抗原的包被浓度为500ng/mL,最适血清稀释度为1∶40。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础。     

猪源支气管败血波氏杆菌百日咳杆菌黏附素基因的原核表达及其抗体检测ELISA方法

[目的]以猪源支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)百日咳杆菌黏附素(PRN)基因的原核表达产物为抗原建立检测PRN抗体的间接ELISA方法.[方法和结果]利用谷胱甘肽-S-转移酶(GST)表达系统对PRN基因在大肠杆菌中进行融合表达.SDS-PAGE和Western blot检测证实该基因获得高效表达,产物易于纯化且具有良好的免疫学活性.通过凝血酶酶切GST-PRN并回收,获得不含GST载体蛋白的PRN蛋白片段.以PRN蛋白片段为抗原建立检测天然PRN抗体的间接ELISA方法.该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性,其敏感性比乳胶凝集试验提高4～128倍,能检测到人工感染14 d后的仔猪血清抗体IgG,对临床送检的1,229份猪血清的检测阳性率为32.7%.ELISA方法对阳性猪场的监测结果预示了保育期仔猪的合群导致猪群大量感染支气管败血波氏杆菌.[结论]该方法具有特异性强、敏感性高、重复性好的特点,可用于猪群PRN抗体水平监测和猪波氏菌病流行病学调查.     

兔支气管败血波氏杆菌单克隆抗体的制备及鉴定

目的建立能稳定分泌抗兔支气管败血波氏杆菌（Bb）的单克隆抗体杂交瘤细胞株,为今后进一步建立该菌的免疫检测技术奠定基础。方法以Bb分离株BLJ05的灭活菌液为免疫原,腹腔免疫BALB/c小鼠,采用常规杂交瘤技术制备Bb单克隆抗体（McAb）,用间接ELISA、Western-blot等方法对McAb特性进行鉴定。结果获得两株能稳定分泌抗Bb单克隆抗体的杂交瘤细胞株,分别命名为A7D5和D6B2,其小鼠腹水抗体效价分别为1∶409600和1∶102400;且不与兔大肠杆菌、多杀性巴氏杆菌、产气荚膜梭菌等兔的常见病原菌反应,特异性强。两株单抗亲和力实验表明A7D5亲和力略高于D6B2。ELISA相加试验表明它们针对相同的抗原表位。结论成功建立了两株能稳定分泌抗兔支气管败血波氏杆菌单克隆抗体的杂交瘤细胞株,效价高、特异性强,为今后建立该菌的免疫检测技术建立奠定了基础。     

兔支气管败血波氏杆菌PCR检测方法的建立及应用

目的建立一种简便、快速、敏感、特异的适用于支气管败血波氏杆菌的PCR检测方法。方法根据兔支气管败血波氏杆菌（Bordetella bronchiseptica）的fim2基因序列设计了一对特异性引物,进行PCR扩增、特异性和敏感性试验,并将其应用于临床样品的检测。结果利用该PCR方法扩增出425bp的目的基因片段,该产物序列与GeneBank上公布的基因序列同源性为100％。特异性试验表明,该方法对大肠埃希氏菌、多杀性巴氏杆菌、魏氏梭菌和金黄色葡萄球菌均无交叉性反应;并且最小可检出菌液浓度为3．6CPU。用该PCR方法检测了从江苏、山东等地采集的146份兔鼻拭子,结果检出支气管败血波氏杆菌阳性92例,阳性率为63．01％。结论建立了快速检测支气管败血波氏杆菌的PCR方法。     

重组百日咳杆菌黏附素蛋白免疫小鼠可完全抵抗支气管败 血波氏杆菌的致死性感染

[目的]通过建立的小鼠呼吸道感染模型评价重组百日咳杆菌黏附素蛋白(GST-PRN)对小鼠的免疫保护效力.[方法和结果]在主动免疫保护试验中,GST-PRN免疫组小鼠能产生较高的PRN抗体水平,在使用3xLD50的支气管败血波氏杆菌HH0809株进行呼吸道气雾攻毒后,其保护率为100%(20/20),但载体蛋白GST和PBS对照组小鼠的存活率仅为15%(3/20)和20%(4/20).在被动免疫保护试验中,腹腔免疫GST-PRN兔抗血清能100%(10/10)保护小鼠抵抗10×LD50的HH0809株的腹腔攻击,但GST兔抗血清和PBS免疫组小鼠的存活率均为0(0/10和0/9).[结论]研究结表明重组PRN蛋白具有良好的免疫学活性,可作为亚单位疫苗或疫苗添加成分.     

支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测

本研究以小鼠为实验动物模型,研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用.本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP,利用SDS-PAGE比较了2株之间差异,采用Western-blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分,采用电洗脱方法获的分子量为68 kD的P13-OMP有效保护抗原成分(OMP68),然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原.抗体动态变化:将清洁级小鼠70只随机分成7组,10只/组,用油乳剂P13的全茵和不同剂量的P13-OMP和OMP68免疫抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体水平并分析抗体动态变化;主动免疫保护试验:将清洁级小鼠80只随机分成8组,10只/组,分别采用制备的油乳剂P13-OMP(OMP25 μg/只)、OMP68(25 μg/只)和P13菌体免疫抗原在免疫10 d后,分别用100 LD50的P11和P13腹腔攻毒,然后统计保护率并分析免疫原所提供的免疫保护力.免疫P13-OMP和OMP68油乳剂抗原后,7 d时,抗体水平开始逐渐上升,42 d时,抗体水平分别达到215.3和214.8,然后逐渐下降,70 d时,抗体水平分别达到29.1和210.2.主动免疫时,P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护,OMP68免疫组分别均得到9/10和10/10的保护,而P13全菌免疫组分别得到4/10和6/10保护,对照组全部死亡.P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用,为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础.     

支气管败血波氏杆菌皮肤坏死毒素的重组表达及其生物学特性

皮肤坏死毒素(DNT)是支气管败血波氏杆菌的主要致病因子之一.通过PCR分段扩增和克隆获得了全长4 356 bp的dnt基因,并利用pET-28a/BL21系统对其进行了融合表达.Western blotting检测结果表明,表达产物具有良好的免疫学活性.使用His-band purification kit纯化试剂盒纯化后,得到纯度为93.2％的融合蛋白His6-DNT.在乳鼠皮肤坏死试验中,表达产物His6-DNT和天然DNT均能导致乳鼠皮肤产生坏死性病变.在乳鼠皮肤坏死阻断试验中,兔抗His6-DNT血清能中和天然DNT使其失去对乳鼠的皮肤坏死毒性.试验结果表明,重组蛋白具有天然DNT的生物学毒性和免疫原性,所产生的抗体具有中和活性.文中所获得的重组DNT蛋白具有良好的生物学活性,为DNT的结构和功能研究奠定基础.     

百日咳杆菌的抗原

鲍特氏菌属的抗原共发现３大类：凝集原,丝状血凝素（ＦＨＡ）及毒素。在凝集原中只有７是百日咳,副百日咳及支气管败血症菌所共有,其他１３种各异。ＦＨＡ与百日咳毒素（ＰＴ）为无细胞苗的主要成分。毒素有５种,最主要的是ＰＴ,其他有不耐热毒素,气管细胞毒（ＴＣＴ）,脂多糖（ＬＰＳ）内毒素及腺嘌呤环化酶毒素（ＡＣＴ）。     

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. I: Protection against homologous stabilate challenge.

Theileria sergenti were isolated from infected erythrocytes by hypotonic lysis, and soluble merozoite antigens were purified by sonication and differential centrifugation. The preparation contained 29, 34, 35 and 105 kD immuno-dominant poly-peptides. The soluble antigens (0.5 mg/ml) were prepared and fortified with Freund's adjuvant. Five month old naive Korean calves were subcutaneously inoculated with the preparation and a booster dose was administered 4 weeks later. Nine weeks after the booster dose, vaccinates and controls were challenged with a homologous stabilate (5.6 x 10(6) RBC/dose, 40% Parasitemia). All animals were monitored for hematocrit, total erythrocyte count, parasitemia and for the specific antibody by Western immunoblot (WB) and indirect immuno-fluorescent antibody (IFA) test. By 18 weeks after vaccination (6 weeks after the challenge), vaccinated cattle had an average IFA titer of 1:10,240 compared with 1:1,280 of the controls. The vaccinates showed negligible change in hematocrit and total RBC count whereas control animals showed significant (p less than 0.05) hematological changes and associated anemia. After vaccination and challenge, the antibody responses demonstrated that vaccination had induced significant production of antibody to the 29 and 35 kD polypeptides. The latter polypeptide was much more strongly recognized by the vaccinated animals, and thus it may be a potential candidate for the vaccine.     

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN‐γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post‐challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD–MPLA than in those that had received rFeSOD‐Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD–MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific‐IFN‐γ responses were significantly stronger in the group vaccinated with rFeSOD–MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.     

Immunogenicity and protective efficacy of Gag/Pol/Env vaccines derived from temporal isolates of SIVmne against cognate virus challenge

BACKGROUND: We used the SIVmne model to examine the relative immunogenicity and protective efficacy of vaccines derived from temporal isolates of lentivirus infection. SIVmne170 is a molecular clone isolated from a pig-tailed macaque 170 weeks after inoculation with SIVmneCL8. METHODS: We immunized pig-tailed macaques with Gag/Pol/Env vaccines derived from CL8 or 170 and examined their protective efficacy against CL8, 170, or chimeras 8/170 and 170/8, containing the 5' or 3' half of the respective parental genomes. RESULTS: As expected, CL8 vaccines protected animals against the CL8, but not the 170 virus. Surprisingly, 170 vaccines not only failed to protect against the 170 virus, but also the less pathogenic CL8. Chimeric virus challenges revealed that the envelope antigen of CL8 represents an important target for protective immunity. CONCLUSIONS: These results underscore the potential importance of targeting transmitted viruses through judicious choice of immunogens from early isolates for vaccine development.     

Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.     

Adaptive evolution of the Bordetella autotransporter pertactin

The virulence factor pertactin is expressed by the closely related pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Pertactin is an autotransporter involved in adherence of Bordetella species to the lung epithelium of mammalian hosts, and it is an important component of most current acellular pertussis vaccines. These three species produce immunologically distinct pertactin molecules, resulting in a lack of cross-protection against B. parapertussis and probably also against B. bronchiseptica. Variation in pertactin is not only inter-specific, but also occurs between isolates from the same species. Knowledge about codons that are under positive selection could facilitate the development of more broadly protective vaccines. Using different nucleotide substitution models, pertactin genes from B. bronchiseptica, B. parapertussis and B. pertussis were compared, and positively selected codons were identified using an empirical Bayesian approach. This approach yielded 15 codons predicted to be under diversifying selection pressure. These results were interpreted in an immunological context and may help in improving future pertussis vaccines.