百日咳毒素和丝状血凝素的纯制和检测现况

介绍了百日咳毒素和丝状血凝素的分离纯化及纯度、活性检定的方法,对影响分离纯化这两种活性物质的因素也作了一些分析     

百日咳丝状血凝素的纯化及初步鉴定

目的从百日咳鲍特菌中提纯百日咳丝状血凝素(filamentous hemagglutinin,FHA),并对其进行初步鉴定。方法采用珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法,从百日咳鲍特菌培养上清中纯化FHA;SDSPAGE电泳和PAGE电泳分析样品纯度,并经斑点免疫印迹法对其进行鉴定。结果纯化样品的纯度达到95%以上,斑点免疫印迹中和FHA单抗有反应斑点,与百日咳毒素(pertussis toxin,PT)单抗无反应斑点。结论珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法可作为一种快速有效的纯化手段提取纯度较高的FHA,有望作为参考品用于百日咳FHA抗原含量检测、抗FHA血清抗体滴度检测及抗原纯度检测。     

无细胞百日咳菌苗纯度和生物学特性研究

本文对无细胞百日咳菌苗的纯度、免疫力和毒性进行了研究。实验证明菌苗中不仅含有百日咳毒素（ＰＴ）和丝状血凝素（ＦＨＡ）,而且还含有百日咳凝集素和百日咳粘着素。菌苗的纯度为５０％－９５．７％,菌苗中ＰＴ和ＦＨＡ的比例为１：１－１：１８。     

四价流感病毒裂解工艺的研究

目的研究流感病毒裂解最佳工艺条件。方法选取BV(NYMC BX-69A)流感病毒毒株,使用BV 1801批纯化液以曲拉通Triton X-100为裂解剂,选取不同浓度、不同裂解时间、不同蛋白质含量等因素,通过每组裂解试验后获得的裂解前后血凝素含量及蛋白质含量,计算血凝素回收率、纯度;40 000倍电镜下观察裂解程度。结果裂解剂曲拉通Triton X-100体积分数为0.5%时,纯度平均值为4.73,血凝素回收率平均值为71.75%;裂解剂曲拉通Triton X-100体积分数为0.7%时,纯度平均值为3.27,血凝素回收率平均值为82.00%;裂解剂曲拉通Triton X-100体积分数为1.0%时,纯度平均值为3.87,血凝素回收率平均值为61.46%。裂解时间为1 h时,纯度平均值为4.17,血凝素回收率平均值为71.75%;裂解时间为2 h时,纯度平均值为3.88,血凝素回收率平均值为82.00%;裂解时间为3 h时,纯度平均值为3.59,血凝素回收率平均值为76.75%。蛋白质浓度为800μg/mL时,纯度平均值为3.80,血凝素回收率平均值为93.00%;蛋白质浓度为1 200μg/mL时,纯度平均值为3.92,血凝素回收率平均值为91.00%;蛋白质浓度为1 600μg/mL时,纯度平均值为4.45,血凝素回收率平均值为77.50%。结论确定了四价流感病毒裂解工艺最佳参数为裂解剂曲拉通Triton X-100体积分数为0.7%,蛋白质含量为800～1 200μg/mL,裂解时间2 h,电镜下观察病毒颗粒完全裂解,血凝素回收率较高,符合工艺标准要求,满足规模化生产。     

百日咳杆菌丝状血凝素

本文扼要介绍了百日咳杆菌丝状血凝素这一有效免疫原的发现过程和提纯方法,并在阐述其物理化学及生物学特性的基础上,就其作为人用疫苗的前景进行了探讨。     

百日咳杆菌69KD外膜蛋白的研究进展

<正>百日咳是由百日咳杆菌引起的一种急性呼吸道传染病,严重威胁婴幼儿健康,对它的控制迄今主要依赖于菌苗预防。目前认为,百日咳杆菌有许多生活学活性成份,包括白细胞增多促进因子(LPF)[又称百日咳毒素(PT)]、丝状血凝素(FHA)、腺苷酸环化酶(AC)、外膜蛋白、脂多糖、不耐热毒素等。其中LPF、FHA、69KD外膜蛋白作为保护性抗原成份,是目前国内外学者研究的热点。     

条斑紫菜丝状体总RNA提取方法比较

目的:为了获得质量较高的条斑紫菜丝状体总RNA,对几种常用提取方法进行研究。方法:以条斑紫菜自由丝状体为材料,比较了用异硫氰酸胍法、CTAB法、SDS/酚法、TRIzol法、RNAplant法提取的RNA的质量和纯度。结果:异硫氰酸胍法提取RNA的成本低,但纯度不高;CTAB法产率较小,且不能完全去除多糖或蛋白质;SDS/酚法未能获得完整的RNA;TRIzol法未能见到5SrRNA条带,且带有杂带;而RNAplant法提取RNA的质量好、纯度高、提取效率高,其D260nm/D280nm值为1.836,经逆转录得到的双链cDNA扩增产物长度在200bp以上。结论:实验结果表明RNAplant法更适于条斑紫菜丝状体总RNA的提取。     

国产层析填料在百日咳毒素和丝状血凝素纯化中的筛选及应用

目的 筛选适合的国产层析填料以满足对组分百日咳疫苗中百日咳毒素(pertussis toxin, PT)与丝状血凝素(filamentous hemagglutinin, FHA)的分离纯化。方法 筛选对PT与FHA结合能力较好且分离度高的国产层析填料，优化纯化工艺，通过3批纯化试验比较国产填料与进口填料的抗原回收率、目的抗原纯度以及对目的抗原的动态结合载量。结果 通过纯化试验筛选到国产填料SP resin-1与MMC resin-1并优化了纯化工艺。3批纯化试验显示，SP resin-1纯化百日咳抗原工艺稳定，FHA的纯度和回收率与Capto SP ImpRes差异无统计学意义(P>0.05);且与Capto SP ImpRes相比，SP resin-1对抗原的动态结合载量更高。3批PT精纯试验显示，国产填料MMC resin-1精纯PT的工艺稳定，PT的纯度和回收率均与Capto MMC差异无统计学意义(P>0.05),并且MMC resin-1对PT的载量高于Capto MMC。结论 试验筛选出纯化PT与FHA的国产填料SP resin-1与MMC resin-1,纯...     

副粘病毒血凝素-神经氨酸酶蛋白结构与功能的研究进展

副粘病毒的血凝素-神经氨酸酶和融合蛋白具有重要的生物学活性,其中前者具有受体识别活性、神经氨酸酶活性和促进融合蛋白的细胞融合作用.本文对近年来血凝素-神经氨酸酶结构和功能方面的研究进展进行了综述.     

腺苷酸环化酶毒素在无细胞百日咳疫苗中的免疫保护效果评价

目的 利用小鼠百日咳感染模型评估在无细胞百日咳疫苗中加入腺苷酸环化酶毒素的C端结构域(RTX751),能否提升无细胞百日咳疫苗的免疫保护效果.方法 ①用腺苷酸环化酶毒素的C端结构域与百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamentous hemagglutinin,FHA)、百日咳黏附素...     

In vitro evaluation of human osteoblast-like cell proliferation and attachment on nanostructured fluoridated hydroxyapatite

The effect of the fluorine content and nano-structure of fluoridated hydroxyapatite (FHA) on human osteoblast-like (HO) cell behavior were investigated. FHA nanopowders and bulk nanostructured FHA, produced via mechanical alloying and two-step sintering, respectively, were used. The cytotoxicity of FHA nanopowders was assessed by MTT. Cell attachment to the surface of the bulk nanostructured FHA was evaluated by culturing of HO cells. Although HO cells proliferated 10 % more in contact with FHA nanopowders compared to culture medium without FHA nanopowders, an increase in the fluorine content of FHA caused a delay in the cell proliferation by about 2 days. Cell attachment on the bulk nanostructured FHA did not change the fluorine content.     

FHA Domain-Mediated DNA Checkpoint Regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9

Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously.     

The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.     

FHA domain-mediated DNA checkpoint regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

The effect of methyl-β-cyclodextrin on the stability of Bordetella pertussis filamentous haemagglutinin

Abstract It has been demonstrated that filamentous haemagglutinin (FHA) purified from Bordetella pertussis is stable on static incubation but is unstable and quickly loses HA activity when incubated with shaking. Methylβcyclodextrin (CD) was found to have a concentration-dependent stabilizing effect on FHA incubated with shaking, suggesting that the ability of CD to enhance yields of FHA in shaken cultures could be wholly or partly due to a stabilizing effect of CD on FHA. However, only weak binding of CD to FHA was demonstrated by an ultrafiltration micropartition method and binding of CD to B. pertussis cells was not related to the presence or absence of FHA on the cell surface.     

Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53

The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human. It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides. Neither the structure nor the ligand of FHA is known. Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9. We herein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730). The structure consists of a beta-sandwich containing two antiparallel beta-sheets and a short, C-terminal alpha-helix. Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains. The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity.     

Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate

The Chfr mitotic checkpoint protein is frequently inactivated in human cancer. We determined the three-dimensional structure of its FHA domain in its native form and in complex with tungstate, an analog of phosphate. The structures revealed a beta sandwich fold similar to the previously determined folds of the Rad53 N- and C-terminal FHA domains, except that the Rad53 domains were monomeric, whereas the Chfr FHA domain crystallized as a segment-swapped dimer. The ability of the Chfr FHA domain to recognize tungstate suggests that it shares the ability with other FHA domains to bind phosphoproteins. Nevertheless, differences in the sequence and structure of the Chfr and Rad53 FHA domains suggest that FHA domains can be divided into families with distinct binding properties.     

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.     

FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea

Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.