共查询到20条相似文献,搜索用时 31 毫秒
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M Kh Gorlina E B Lapina A V Rodionov E K Fan'kovskaia 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1990,(3):61-65
The study of protective cross-reacting antigenic preparations isolated from meningococci of groups A and C in the blot immunoassay has shown the presence of a group of proteins with a molecular weight ranging from 23 to 31 KD and common for 8 tested serological groups of meningococci, gonococci and 4 nonpathogenic Neisseria species. The possible role of these structures as common Neisseria antigen in the formation of natural resistance to meningococcal infection is discussed. 相似文献
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Gupta PK Kurupati RK Chandra H Gaur R Tandon V Singh Y Maithal K 《Biochemical and biophysical research communications》2003,311(1):229-232
Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state. 相似文献
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Chalton DA Kelly IF McGregor A Ridley H Watkinson A Miller J Lakey JH 《Archives of biochemistry and biophysics》2007,465(1):1-10
Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum. 相似文献
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Isolation of protective antigen from Bordetella pertussis 总被引:5,自引:0,他引:5
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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
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Recombinant protective antigen (rPA), expressed by Bacillus subtilis WB600 (pPA101), has been purified to homogeneity and the protective efficacy against a Bacillus anthracis challenge has been investigated. rPA was fractionated from culture supernatant fluid by ammonium sulphate, followed by anion exchange chromatography using DEAE Streamline™, anion-exchange chromatography on FPLC MonoQ HR 10/10 and finally, gel filtration chromatography on FPLC Superose 12 HR 10/30, to yield 7 mg rPA per litre of culture. The protective efficacy of rPA against an airborne challenge with the AMES strain of B. anthracis was determined in the presence of the adjuvants, alhydrogel and Ribi, and compared to that achieved by the current UK human vaccine in guinea pigs. rPA combined with the Ribi adjuvant was found to provide 100% protection against challenge. 相似文献
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Brodzik R Spitsin S Golovkin M Bandurska K Portocarrero C Okulicz M Steplewski Z Koprowski H 《Cancer immunology, immunotherapy : CII》2008,57(3):317-323
Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence
and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields
using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic
and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived
antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal
carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility
of producing anti-cancer recombinant vaccines using plant expression systems. 相似文献
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PA(63), the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH >/=8.0, this "prepore" dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH =7 (or with beta-octylglucoside at pH 8.0) caused it to convert to an SDS-resistant pore-like form. Transition to this form involved major changes in the conformation of loop 2 of domain 2 (D2L2), as evidenced by (i) occlusion of a chymotrypsin site within D2L2 and (ii) excimer formation by pyrene groups linked to N306C within this loop. The pore-like form retained the capacity to bind anthrax toxin A moieties and cell surface receptors, but was unable to form pores in membranes or mediate translocation. Mutant PA(63) in which D2L2 had been deleted was inactive in pore formation and translocation but, like the prepore, was capable of forming heptamers that converted to an SDS-resistant form under acidic conditions. Our findings support a model of pore formation in which the D2L2 loops move to the membrane-proximal face of the heptamer and interact to form a 14-strand transmembrane beta-barrel. Concomitantly, domain 2 undergoes a major conformational rearrangement, independent of D2L2, that renders the heptamer resistant to dissociation by SDS. These results provide a basis for further exploration of the role of PA(63) in translocation of anthrax toxin's enzymic moieties across membranes. 相似文献
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Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax. 相似文献
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The superficial protective antigen of R. prowazeki 总被引:5,自引:0,他引:5
H M Golinevitch Z A Voronova 《Journal of hygiene, epidemiology, microbiology, and immunology》1968,12(4):413-419
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Sun J Vernier G Wigelsworth DJ Collier RJ 《The Journal of biological chemistry》2007,282(2):1059-1065
Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore. 相似文献
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Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus 总被引:11,自引:0,他引:11
P Willadsen G A Riding R V McKenna D H Kemp R L Tellam J N Nielsen J Lahnstein G S Cobon J M Gough 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(4):1346-1351
Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum. 相似文献