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1.
N. I. Mikshis O. M. Kudryavtseva D. V. Shulepov A. Yu. Goncharova M. F. Bolotnikova L. V. Novikova Yu. A. Popov V. V. Kutyrev 《Applied Biochemistry and Microbiology》2011,47(7):667-673
An asporogenic recombinant strain Bacillus anthracis 55ΔTPA-1(Spo−) producing anthrax protective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon pUB110PA-1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity.
The level of PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than
the values determined for vaccine strains B. anthracis STI-1 and B. anthracis 55. The strain preserves asporogenicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically
active PA was isolated from the constructed strain B. anthracis 55ΔTPA-1(Spo−). Double immunization of rabbits with 50 μg of the purified recombinant product provides their 100% protection from infection
with 50 LD50 of a highly virulent anthrax strain. 相似文献
2.
Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen 总被引:8,自引:5,他引:3
Stéphane Mesnage Evelyne Tosi-Couture Michèle Mock Pierre Gounon Agnès Fouet 《Molecular microbiology》1997,23(6):1147-1155
Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology’motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen. 相似文献
3.
Uchida M Harada T Enkhtuya J Kusumoto A Kobayashi Y Chiba S Shyaka A Kawamoto K 《Biochemical and biophysical research communications》2012,421(2):323-328
Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA. 相似文献
4.
Ramirez DM Leppla SH Schneerson R Shiloach J 《Journal of industrial microbiology & biotechnology》2002,28(4):232-238
The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a
molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations
found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine.
The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These
proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which
lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient
production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors
composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time
was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed
by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation,
microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after
purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of
formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239
Received 28 August 2001/ Accepted in revised form 20 December 2001 相似文献
5.
将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因.以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B.anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌.然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定.最终建立了Cre-LoxP系统在B.anthracis中的应用方法,并成功敲除eag基因. 相似文献
6.
Fermentation, Purification, and Characterization of Protective Antigen from a Recombinant, Avirulent Strain of Bacillus anthracis 下载免费PDF全文
Bacillus anthracis, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an Mr 83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of B. anthracis and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values. 相似文献
7.
N. Ghosh G. Gupta M. Boopathi V. Pal A. K. Singh N. Gopalan A. K. Goel 《Indian journal of microbiology》2013,53(1):48-55
Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation. 相似文献
8.
Fatemeh Vahedi Fatemeh Khatami Ali Es-haghi 《World journal of microbiology & biotechnology》2011,27(2):253-257
The recombinant plasmid pET21 + PA that has been deposited at Genbank with accession number EF550209 was constructed by inserting
the 1,700-bp PA (protective antigen of Bacillus anthracis) recombinant gene into Xho I/Hind Ш sites of the pET21b + vector under the control of the T7 promoter for highly expressing PA. pET21 + PA was cloned into
Escherichia coli BL21 strain. The high activity of T7 RNA polymerase could make a powerful expression system for high-level expression of
the recombinant proteins. However, during the large-scale production of recombinant proteins, the productivity of a fermentation
process is directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this
study, we studied the effects of various culture conditions on the plasmid stability and target protein yield including antibiotic
concentrations, the time of induction by IPTG, and the number of successive cultures. The results indicated that the plasmid
pET21 + PA is completely stable after the fiftieth generation. Loss of plasmid and structural change were not detected but
the yield of protein production was decreased by about 10% in generation 50. These data would be useful for the industrial
production of the recombinant PA vaccine and other recombinant proteins. 相似文献
9.
The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase
the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional
treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the
Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed
that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T1 transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T2 generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only.
These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants. 相似文献
10.
Baillie L Hebdon R Flick-Smith H Williamson D 《FEMS immunology and medical microbiology》2003,36(1-2):83-86
The UK human anthrax vaccine consists of the alum-precipitated culture supernatant of Bacillus anthracis Sterne. In addition to protective antigen (PA), the key immunogen, the vaccine also contains a number of other bacteria- and media-derived proteins. These proteins may contribute to the transient side effects experienced by some individuals and could influence the development of the PA-specific immune response. Bacterial cell-wall components have been shown to be potent immunomodulators. B. anthracis expresses two S-layer proteins, EA1 and Sap, which have been demonstrated to be immunogenic in animal studies. These are also immunogenic in man so that convalescent and post-immunisation sera contain specific antibodies to Ea1, and to a lesser extent, to Sap. To determine if these proteins are capable of modifying the protective immune response to PA, A/J mice were immunised with equivalent amounts of recombinant PA and S-layer proteins in the presence of alhydrogel. IgG isotype profiles were determined and the animals were subsequently challenged with spores of B. anthracis STI. The results suggest that there was no significant shift in IgG isotype profile and that the presence of the S-layer proteins did not adversely affect the protective immune response induced by PA. 相似文献
11.
Dian-Bing Wang Ruifu Yang Zhi-Ping Zhang Li-Jun Bi Xiang-Yu You Hong-Ping Wei Ya-Feng Zhou Ziniu Yu Xian-En Zhang 《PloS one》2009,4(11)
Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores. 相似文献
12.
Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S‐layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time‐to‐death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames. 相似文献
13.
Fatemeh Vahedi Jennelle Kyd Gholamreza Moazeni Jula Monavvar Afzalaghaeei Mahdi Kianizadeh Mahmoud Mahmoudi 《World journal of microbiology & biotechnology》2008,24(10):2171-2178
The protective antigen (PA) of Bacillus anthracis (B. anthracis) is a potent immunogen and a candidate subunit vaccine. To address the question whether antibodies raised against PA following
injection of pcDNA3.1+PA plasmid, encoding PA, can protect against virulent B. anthracis two different regimens of PA based vaccines (DNA and live spore) were used. The groups of BALB/c mice that received live
spores of the Sterne strain, naked pcDNA3.1 and naked pcDNA3.1+PA were compared to control groups. All groups were injected
three times with 30-day intervals. Two weeks after the last immunization, all mice were subjected to challenge with a pathogenic
strain of B.
anthracis (C2). Blood samples were taken before each injection and challenge. Evaluation of the sera by ELISA method showed that DNA
immunization using pcDNA3.1+PA plasmid resulted in an antibody profile representative of a mixed Th1 and Th2 response, with
a skewing to a Th1 response. The group which received the naked pcDNA3.1+PA had a survival rate of >80%. This challenge assay
revealed that antibodies raised following DNA vaccination against PA can confer strong protection, and resistance against
virulent species of B. anthracis. 相似文献
14.
《Microbes and infection / Institut Pasteur》2009,11(3):424-428
The possibility of expressing a homologous antigen and a heterologous antigen simultaneously in an attenuated Brucella melitensis strain was investigated. The Brucella wboA gene encoding a mannosyltransferase involved in biosynthesis of lipopolysaccharide O-antigen, and the Bacillus anthracis pag gene encoding the protective antigen (PA) were cloned into plasmid pBBR4MCS. The resulting plasmid was introduced into O-antigen deficient B. melitensis strain WRRP1 to produce strain WRSPA. Strain WRSPA produced O-antigen and a series of PA products, induced protection in BALB/c mice against challenge with B. melitensis strain 16M, but failed to protect A/J mice against challenge with B. anthracis Sterne strain. 相似文献
15.
Shahid S Park JH Lee HT Kim SJ Kim JC Kim SH Han DM Jeon DI Jung KH Chai YG 《Journal of microbiology (Seoul, Korea)》2010,48(6):771-777
Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite
anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment.
To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4–7), followed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting
using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins
were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two
plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors. 相似文献
16.
Jian Chen Chang‐Hyuk Kwon Lu Lin Yanjiao Li Luis F. Parada 《Genesis (New York, N.Y. : 2000)》2009,47(2):122-131
To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ERT2). In the background of the Cre reporter mouse strain Rosa26lacZ, we show that the fusion CreERT2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc. 相似文献
17.
I. Yu. Egorova T. A. Sevskii Yu. O. Selyaninov 《Applied Biochemistry and Microbiology》2016,52(8):733-738
The results of a study on the immunobiological properties of a new, naturally attenuated, unencapsulated Bacillus anthracis 363/11 strain in comparison with the properties of the anthrax vaccine strain 55-VNIIVVIM, which is currently used in Russia and post-Soviet states, are presented in the article. Some differences in the phenotypes of the aforementioned strains are shown. The new strain caused lysis of sheep erythrocytes by α- but not by β-type; the strain had many more active proteinases and synthesized protocatechuic acid (PCA), in contrast to the vaccine strain. It was ascertained that immunization of animals by the new strain protects them from death after infection by field isolates of B. anthracis, while the 55-VNIIVVIM strain does not create this immunity. The data indicate that the new strain can be used to develop and improve anthrax prevention. 相似文献
18.
Gaobing Wu Chunfang Feng Yuzhi Hong Aizhen Guo Sha Cao Junli Dong Ling Lin Ziduo Liu 《Applied microbiology and biotechnology》2010,87(2):609-616
The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and
the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related
research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically
enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to
high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover,
we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study.
Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant
may be useful in designing new antitoxin for anthrax prophylaxis and therapy. 相似文献
19.
通过比较四种品系小鼠对炭疽芽胞杆菌(Bacillus anthracis)(简称炭疽杆菌)弱毒株芽胞的敏感性,确定炭疽杆菌弱毒株芽胞攻毒合适的动物模型。采用炭疽杆菌弱毒株A16Q1(pXO1-、pXO2+)和A16PI2(pXO1+、pXO2-)的芽胞对四种品系小鼠(DBA/2、KM、ICR和BALB/c)进行腹腔攻毒,记录小鼠死亡时间,计算LD50、绘制存活曲线并统计分析。运用较敏感的KM小鼠研究不同canSNP基因型毒素缺陷株(含pXO2拷贝数不同)芽胞的毒力差异。利用更为敏感的DBA/2小鼠评价S-层蛋白BA3338对荚膜缺陷株芽胞毒力的影响。结果表明,在四种品系小鼠中,毒素缺陷株芽胞的毒力均高于荚膜缺陷株芽胞的毒力。DBA/2小鼠对炭疽杆菌弱毒株芽胞的剂量依赖关系最好,最为敏感,其次是KM小鼠,而ICR小鼠和BALB/c小鼠对炭疽杆菌弱毒株芽胞不敏感。确定了DBA/2小鼠和KM小鼠在炭疽杆菌弱毒株芽胞研究中的适用性。使用KM小鼠评价了不同canSNP基因型炭疽杆菌芽胞的毒力差异,结果表明,不同canSNP基因型炭疽杆菌由于所含pXO2质粒拷贝数的差异导致芽胞的毒力不同。使用DBA/2小鼠评价了S-层蛋白BA3338缺失对炭疽杆菌芽胞毒力的影响,表明BA3338基因的缺失导致炭疽杆菌芽胞毒力降低。 相似文献
20.
Responding to the threat of bioterrorism: a microbial ecology perspective – the case of anthrax 总被引:2,自引:0,他引:2
R. Atlas 《International microbiology》2002,5(4):161-167
Anthrax is a disease of herbivores caused by the gram-positive bacterium Bacillus anthracis. It can affect cattle, sheep, swine, horses and various species of wildlife. The routes for the spread among wildlife are
reviewed. There are three kinds of human anthrax – inhalation, cutaneous, and intestinal anthrax – which differ in their routes
of infection and outcomes. In the United States, confirmation of cases is made by the isolation of B. anthracis and by biochemical tests. Vaccination is not recommended for the general public; civilians who should be vaccinated include
those who, in their work places, come in contact with products potentially contaminated with B. anthracis spores, and people engaged in research or diagnostic activities. After September 11, 2001, there were bioterrorism anthrax
attacks in the United States: anthrax-laced letters sent to multiple locations were the source of infectious B. anthracis. The US Postal Service issued recommendations to prevent the danger of hazardous exposure to the bacterium. B. anthracis spores can spread easily and persist for very long times, which makes decontamination of buildings very difficult. Early
detection, rapid diagnosis, and well-coordinated public health response are the key to minimizing casualties. The US Government
is seeking new ways to deter bioterrorism, including a tighter control of research on infectious agents, even though pathogens
such as B. anthracis are widely spread in nature and easy to grow. It is necessary to define the boundary between defensive and offensive biological
weapons research. Deterring bioterrorism should not restrict critical scientific research.
Electronic Publication 相似文献