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1.
Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.  相似文献   

2.
Abstract Cell envelope preparations of Treponema hyodysenteriae (strain CN 8368) were examined using biochemical and immunochemical methods. Several major polypeptides were detected with molecular weight between 24-kDa and 45-kDa. The majority of these polypeptides were recognised by serum from a pig vaccinated with an experimental whole-cell T. hyodysenteriae vaccine and hyperimmune anti- T. hyodysenteriae rabbit sera. Immune electron microscopy confirmed that the major antigens detected were associated with the cell envelope. Triton X-100, in the presence of EDTA, completely solubilised a polypeptide with an approximate molecular weight of 36-kDa. Antibodies to this polypeptide were not absorbed by whole T. innocens cells.  相似文献   

3.
In order to examine the relationship between the intermediate filaments from Purkinje fibres of the cow heart conduction system and five proposed subclasses of mammalian intermediate filaments, the gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) has been used to examine the specificity and crossreactivity of our antibodies against the Purkinje fibre intermediate filament protein, skeletin. Bovine tissues known to contain intermediate filaments of the five main subclasses were examined with antiskeletin and with preimmune serum and the specific antiserum absorbed with pure skeletin as controls. The antibodies raised against Purkinje fibre skeletin reacted with all three polypeptides of the "neurofilament triplet", with glial fibrillary acidic protein (GFAP), with smooth muscle desmin and also slightly with some prekeratin subunits and with endothelial vimentin. From studies with monoclonal antibodies and amino acid sequencing, certain regions of all intermediate filaments are suggested to be structurally related. Here we show that Purkinje fibre skeletin seems to share antigenic determinants with the proposed five main classes of intermediate filaments. Our antibody is the first carefully controlled experimentally induced antibody having such properties. This might be due to the special attributes of the intermediate filament system in Purkinje fibres, which themselves have unique properties.  相似文献   

4.
Alcoholic hyalin is an hepatocellular aggregate composed of filaments apparently related to the prekeratin intermediate filament subclass. The relationship between these two filament preparations was determined immunochemically using guinea pig antisera derived against alcoholic hyalin, prekeratin, and major prekeratin polypeptides. Immunocrossreactivities were determined using sensitive solid-phase enzyme-immunoassays. These assays indicated that antisera derived against a given filament preparation reacted 10–1000 times better with that preparation than with the other system. The nature of crossreactive meterial was determined using antisera derived against the larger prekeratin polypeptides (Mr 61,000 and 51,000). When tested against these two antisera, alcoholic hyalin appeared to react better with the serum derived against the larger prekeratin component. Moreover, anti-alcoholic hyalin antiserum bound four to five times better to the 61,000 dalton component than to the 51,000 dalton polypeptide in the enzyme-immunoassay. Our results indicate that antigenic determinants related to prekeratin can be detected in alcoholic hyalin, but that these determinants are present in relatively low concentrations in purified alcoholic hyalin. In addition, it appears that the relative concentrations of prekeratin components in alcoholic hyalin do not reflect those in purified prekeratin.  相似文献   

5.
Intermediate filaments in nervous tissues   总被引:59,自引:30,他引:29  
Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.  相似文献   

6.
R Vrijsen  B Rombaut    A Boeye 《Journal of virology》1984,49(3):1002-1004
Quantitative data are presented on (i) the intertypic cross-reactions of polyclonal, guinea pig antibodies directed against the N or H antigen of type 1 poliovirus and (ii) a set of five nonneutralizing, mouse hybridoma antibodies raised against N antigen or a mixture of capsid polypeptides VP1, VP2, and VP3. Three of these antibodies recognize H antigen and VP1, the fourth H antigen only, and the fifth VP3 only. The antibodies recognize either only homotypic antigens or the antigens of the three serotypes.  相似文献   

7.
Abstract Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent 'landmark' polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane-associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa.  相似文献   

8.
Rabbit antisera were produced against whole cells and against highly purified axial filaments of Treponema zuelzerae. Both types of sera react with axial filaments. Antisera against whole cells immobilize the organism; antisera against axial filaments do not. The immobilizing activity of anticell sera could be neutralized by preabsorption with whole cells but not by preabsorption with axial filaments. Preabsorption with axial filaments did, however, abolish the ability of anticell sera to react with axial filaments. Anticell sera also react with the outer cell envelope; this ability was not affected by preabsorption with axial filaments. The data show that antibody directed at something other than axial filaments causes immobilization of T. zuelzerae. The results do not exclude the possibility that the axial filament is the organelle of motility. Titration of the immobilizing activity of anticell sera by adding a constant amount of motile cells to serial dilutions of serum gave two zones of maximum immobilization, one with undiluted serum and one at higher dilutions. For a fixed amount of antiserum, increasing the numbers of cells in the titration almost, but not quite, abolished the zone phenomenon. This phenomenon appears to result from the presence of two kinds of immobilizing antibodies in anticell serum. One of these belongs to the IgG class of immunoglobulins. The other has not been identified but is present in a serum globulin fraction which contains IgM. At intermediate concentrations, the "IgM fraction" inhibits the immobilizing activity of IgG without itself causing immobilization.  相似文献   

9.
Antibody decoration of neurofilaments   总被引:36,自引:18,他引:18       下载免费PDF全文
We have decorated neurofilaments with antibodies against three polypeptides (designated here as H [mol wt = 195,000], 45[mol wt = 145,000], and 46[mol wt = 73,000]) in an effort to understand the arrangement of these polypeptides within neurofilaments. The three polypeptides were purified and antibodies were raised against each. The cross-reactivity of the antibodies suggested that each polypeptide contains both shared and unique antigenic determinants. The differential reactivities of each antibody preparation were enhanced by adsorption with the two heterologous polypeptides, and the resulting preparations were used to decorate purified neurofilaments, which were then negatively stained and examined in an electron microscope. The appearance of the antibody-decorated structures led to the following conclusions: All three polypeptides are physically associated with the same neurofilament. However, the disposition of H and 46 within a filament is different; 46 antigens appear to be associated with a "central core" of the filament, whereas H antigens compose a structure more loosely and peripherally attached to the central core and periodically arranged along its axis. The antibody-decorated H- containing structure assumes variable configurations; in some cases it appears asa bridge connecting two filaments; in other cases it appears as a helix wrapping the central core with a period of approximately 1,000 A and an apparent unit length of approximately 1.5 periods. These configurations suggest several functional implications, including the possibility that H is a component of the cross-bridges observed between filaments in situ. We also note that the central core-helix relationship could be used in the design of an intracellular transport motor.  相似文献   

10.
Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.  相似文献   

11.
A rapid, new procedure for the isolation of intact tonofilaments from newborn rat skins is described. The filament preparations show two major protein subunits on SDS-PAGE with molecular weights of 58000 and 66000 D. An antiserum prepared against the 58000 D protein reacted specifically with the tonofilament preparation, but not with the protein subunits of neurofilaments, glial filaments, tubulin or actin. This specificity is confirmed by indirect immunofluorescence: anti-P58 reacts with the epidermis, whereas antisera against the neurofilament or glial filament proteins and anti-tubulin do not. These data suggest that epidermal filaments represent a class of intermediate filaments distinct from either glial filaments or neurofilaments.  相似文献   

12.
Mice fed griseofulvin, an antibiotic with antimicrotubular activity, formed hepatocellular aggregates of intermediate filaments, which resembled those associated with human alcoholic liver disease. These aggregates, termed Mallory bodies, were isolated from both human and mouse liver and the composition of these structures compared. Electrophoretic analysis indicated that the mouse filaments were composed of four major polypeptides (51,000, 47,000, 37,000, and 36,000 daltons). Human Mallory bodies possessed a similar number of components but of different molecular weights (56,000, 51,000, 50,000, and 38,000 daltons). Guinea pig antisera prepared against both whole human Mallory bodies and the major human polypeptide (56,000 daltons) crossreacted with mouse Mallory body material in both immunochemical and immunocytochemical systems. Our findings suggest that the two filament systems possess similar biochemical and immunological properties.  相似文献   

13.
In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite.  相似文献   

14.
Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.  相似文献   

15.
Hepatocytes of mammalian liver are known to contain intermediate-sized filaments of tonofilament morphology. Unlike many other epithelial cells, including cultured hepatocytes and hepatoma cells, hepatocytes present in normal liver tissue have been reported not to react, in significant intensity, with various preparations of antibodies to human and bovine epidermal prekeratin [2,6]. We have therefore examined, by biochemical and immunological methods, the cytoskeletal composition of hepatocytes grown in the body.Cytoskeletal preparations from hepatocytes of mouse and rat liver tissue resistant to high salt buffer and Triton X-100 are enriched in tangles of intermediate filaments and contain, besides some residual microfilamentous actin, a characteristic set of polypeptides. One- and two-dimensional gel electrophoresis reveals the presence of two major cytokeratin components, which appear as ‘pairs’ of isoelectric variants (component A, Mr 55 000, apparent pI values, 6.40 and 6.45; component D, Mr 49000, apparent pI values 5.43 and 5.38), and five minor components (Mr range from 41000 to 53 000), most of them also as ‘pairs’ of polypeptides slightly different in isoelectric pH value. These polypeptide patterns are very similar in mouse and rat liver although some minor but significant differences have been noted between the two species. The polypeptide patterns of liver cytoskeletons are also similar to—but clearly not identical with—the cytoskeletal protein patterns observed in other epithelial tissues and cells, including various lines of cultured rat hepatocytes and hepatoma cells.Guinea pig antibodies raised against individual cytokeratin proteins of mouse liver and against certain prekeratin polypeptides present in desmosome-attached tonofilaments of bovine muzzle are described which differ from previously described prekeratin antibodies. These prekeratin antibodies not only react with filament bundles of the prekeratin type present in many cultured epithelial cells (e.g. murine HEL, human HeLa, rat kangaroo PtK2) and various epithelial tissues, but also allow the detection of the cytokeratin components present in parenchymal cells of liver and pancreas of various species, man included. Immunofluorescence microscopy on frozen sections of liver using these antibodies reveals a novel structure, i.e. a three-dimensional filament meshwork extending throughout the whole cytoplasm of the hepatocyte, with higher intensity of staining in pericanalicular regions.The results show that parenchymal cells of normal liver and pancreas contain filaments of the cytokeratin type that are related to but not identical with epidermal prekeratin. The hepatocyte filaments appear to be different from prekeratin-type filaments present in epidermis and several other epithelial cells, both in some antigenic determinants exposed and in polypeptide composition. Our findings support the concept of the existence of a family of intermediate filament proteins, cytokeratins, containing many different polypeptides that are expressed in different epithelial cells in certain characteristic subsets in a cell type-specific mode.  相似文献   

16.
The C-termini of Spidroins produced in the major and minor ampullate glands of spiders are highly conserved. Despite this conservation, no corresponding peptides have been identified in the spinning dopes or the silk filaments so far. To prove their presence or absence, polyclonal antibodies derived against fusion proteins containing the conserved C-terminal regions of both Spidroin 1 and 2 from the spider Nephila clavipes were generated. The antibodies reacted with high molecular weight polypeptides of the corresponding gland extracts and solubilized major ampullate filament and in addition to filament cross-sections. This demonstrates the existence of C-terminal specific peptides in the spinning dope and the mature Spidroins. Both the fusion proteins as well as the proteins contained within the gland lumen showed a reduction in their size under reducing conditions indicating the presence of disulfide bonds. Their high conservation and the biochemical data suggest crucial roles the C-termini play in the formation and/or structure of the corresponding silk filaments.  相似文献   

17.
Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.  相似文献   

18.
Jürgen Voigt 《Planta》1988,173(3):373-384
Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDa kilodalton - Mr relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol  相似文献   

19.
Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [125I] and precipitation of the [125I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.  相似文献   

20.
After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.  相似文献   

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