• Title/Summary/Keyword: outer membrane protein H

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Immunogenicity of Recombinant Outer Membrane Protein H from Pasteurella multocida (재조합 파스튜렐라 외막 단백질 H의 면역원성 검정)

  • Lee Jeong-Min
    • Microbiology and Biotechnology Letters
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    • v.34 no.3
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    • pp.273-277
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    • 2006
  • To investigate the antigenicity and protective immunity of outer membrane protein H (OmpH) in Pasteurella multocida D:4, the recombinant OmpH protein was produced in Escherichia coli. The truncated and Trx-fused form of recombinant OmpH (53 kDa) was purified, and used as an antigen in the immunization and challenge experiment. The immunized mice with the recombinant OmpH produced a high-titer antibody, and had protective immunity against P. multocida as same level as the mice immunized with formalin-killed whole cell.

Outer Membrane Protein H for Protective Immunity Against Pasteurella multocida

  • Lee, Jeong-Min;Kim, Young-Bong;Kwon, Moo-Sik
    • Journal of Microbiology
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    • v.45 no.2
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    • pp.179-184
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    • 2007
  • Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

Protective Antibodies and Immunity elicited by Immunization with Outer Membrane Protein H of Pasteurella multocida in Mice (Pasteurella multocida의 외막 단백질 H에 의해 유도되는 방어적 항체와 면역)

  • Kwon, Moo-Sik;Kim, Young-Bong;Lee, Jeong-Min
    • Korean Journal of Microbiology
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    • v.43 no.1
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    • pp.7-13
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    • 2007
  • Pasteurella multocida is one of the important animal pathogen causing widespread infections in various domestic animals. In swine, it causes severe respiratory diseases such as atrophic rhinitis and pneumonic pasteurellosis. To develop the efficient subunit vaccine against swine atrophic rhinitis, we investigated protective antibodies and humoral immunity of outer membrane protein H (OmpH) which is one of the major outer membrane proteins in P. multocida. Outer membrane fraction of P. multocida was immunologically detectable using antisera from both mice groups vaccinated by formalin-killed whole cells and by commercial vaccine. The expression vector for production of recombinant OmpH was constructed and the recombinant OmpH was expressed and purified from E. coli. Recombinant OmpH showed high antigenic and immunogenic properties in mice vaccination and ELISA with antisera.

Molecular Cloning and Characterization of the Gene for Outer Membrane Protein H in a Pasteurella multocida (D:4) Isolate from Pigs with Atrophic Rhinitis Symptoms in Korea

  • LEE, JEONG-MIN;KANG, SEO-YOUNG;PARK, SHIN-IN;WOO, HEE-JONG;KWON, MOO-SIK
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1343-1349
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    • 2004
  • A native strain of Pasteurella multocida was isolated from pigs suffering from severe atrophic rhinitis at domestic farms in Gyeonggi Province, Korea, and was identified as capsular serogroup 'D' and somatic serotype '4' by disc diffusion decapsulation and gel diffusion precipitation tests, respectively. The P. multocida (D:4) induced atrophic rhinitis in healthy pigs by the secondary infection. The gene for outer membrane protein H (ompH) of P. multocida (D:4) was cloned in Escherichia coli DH5$\alpha$ by PCR. The open reading frame of the ompH was composed of 1,023 bp, possibly encoding a protein with 341 amino acid residues containing a signal peptide of 20 amino acids at N-terminus, and the gene product with molecular mass of ca. 38 kDa was identified by SDS-PAGE. Hydropathy profiles indicated that there are two variable domains in the OmpH. To express the ompH in E. coli, the gene was manipulated in various ways. Expression of the truncated as well as full-length forms of the recombinant OmpH was fatal to the host E. coli BL21 (DE3). However, the truncated OmpH fused with GST was consecutively expressed in E. coli DH5$\alpha$. A large quantity of the fused polypeptide was purified through GST-affinity chromatography.

Effects of Egg Yolk Antibodies Produced in Response to Different Antigenic Fractions of E. coli O157:H7 on E. coli Suppression

  • Chae, H.S.;Singh, N.K.;Ahn, C.N.;Yoo, Y.M.;Jeong, S.G.;Ham, J.S.;Kim, D.W.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.11
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    • pp.1665-1670
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    • 2006
  • The objective of this research was to provide the characterization and method for producing anti-E. coli O157:H7 antibodies in egg-laying hens and to determine if the antibody can restrain the proliferation of E. coli O157:H7 in-vitro. Selected antigenic fractions (whole cell, outer membrane protein and lipopolysaccharide (LPS)) from E. coli O157:H7 were injected to hens in order to produce anti-E. coli O157:H7 antibodies. The immune response and the egg yolk antibodies of laying hens against the whole cell, outer membrane protein and LPS antigens were monitored by ELISA. The level of antibodies against whole cell antigen monitored through ELISA sharply increased after the initial immunization, and it was found to be maximum on day 49 however, the level was maintained up to day 70. Antibodies (5 mg/ml) directed against the whole cell inhibited E. coli proliferation 10-13 times more than outer membrane protein or LPS. The antibody response against the whole cell antigens appeared to have higher activity in restraining the proliferation of E. coli O157:H7 than antibody against outer membrane protein or LPS. Results reflected that increasing the IgY's in the egg yolk could prevent greater economic losses due to human and animal health from pathogenic bacteria i.e. E. coli O157:H7.

경구투여 백신 후보물질로서의 Helicobacter pylori 외막 단백질의 조사

  • 박형배;최태부
    • Microbiology and Biotechnology Letters
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    • v.25 no.2
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    • pp.129-136
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    • 1997
  • Helicobacter pylori is a spiral-shaped, microaerophilic human gastric pathogen causing chronic-active gastritis in association with duodenal ulcer and gastric cancer. To investigate the possibility of H. pylori outer membrane proteins (OMPS) as the oral vaccine antigens, sarcosine-insoluble outer membrane fraction has been prepared from H. pylori NCTC 11637. The major OMPs having apparent molecular masses of 62 kDa, 54 kDa and 33 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which were identified as urease B subunit (UreB), heat shock protein (Hsp54 kDa) and urease A subunit (UreA), respectively. Minor protein bands of 57 kDa, 52 kDa, 40 kDa, 36 kDa and 31 kDa were also observed. The antigenicity of H. pylori OMPs and antigenic cross-reactivity among the strains were determined by immunoblot analysis using anti-H. pylori OMPs antisera or intestinal lavage solutions. The results showed that UreB, Hsp54 kDa, UreA and 40 kDa proteins vigorously stimulated mucosal immune response rather than systemic immunity. From this results, these proteins seemed to be useful as the antigen candidates for the oral vaccine. The immunoblotting results with surface proteins from eight isolated H. pylori strains were similar to that of H. pylori NCTC 11637. The IgA which had been arised from oral administration of H. pylori OMPs, was able to bind H. pylori whole-cells.

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Molecular Cloning and Expression of a Gene for Outer Membrane Protein H in Pasteurella multocida (A:3) : Production of Antisera against the OmpH (파스튜렐라 (A:3)외막 단백질 H의 유전자 클론닝$\cdot$발현 및 면역혈청 생산)

  • Kim Younghwan;Hwang Heon;Lee Sukchan;Park Eun-Seok;Yoo Sun-Dong;Lee Jeongmin;Yang Joo-Sung;Kwon MooSik
    • Microbiology and Biotechnology Letters
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    • v.33 no.4
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    • pp.274-280
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    • 2005
  • Pasteurella multocida is known to cause widespread infections in husbandry. To induce homologous and heterologous immunity against the infections, outer membrane proteins (OMPs) in the envelope of P. multocida are thought to be attractive vaccine candidates. Outer membrane protein H is considered as the major component of OMPs. In this study, a gene for OmpH was isolated from pathogenic P. multocida serogroup A. The gene was composed of 1,047 nucleotides coding 348 amino acids with signal peptide of 20 amino acids. The amino acid composition showed about 80 to 98 per cent sequence homologies among other 10 strains of P. multocida serogroup A, reported so far. A recombinant ompH, from which signal peptide was truncated, was generated using pRSET A to name 'pRSET A/OmpH-F2'. The pRSET A/OmpH-F2 was well expressed in E. coli BL21(DE3). The truncated OmpH was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Its molecular weight was registered to be 40 kDa on SDS-PAGE gel. In order to generate immunesera against the OmpH, 50 ug of the protein was intraperitoneally injected into mice three times. The anti-OmpH immuneserum recognized about $5{\times}10^{-2}$ng quantity of the purified OmpH. It can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (Serogroup A).

Electrophoretical Analysis of 36-Kilodalton Outer Membrane Protein of Vibrio vulnificus ATCC 27562

  • Moon-Soo Heo;Cho-Rok Jung
    • Journal of Life Science
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    • v.9 no.1
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    • pp.35-39
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    • 1999
  • Elecrophoreticl analysis of a 36 kDa protein was runned by SDS-PAGE, isoelectric focusing (IEF) and two dimensional electrophoresis pattern. Major 36 kDa and 25, 46, 48, 66 kDa protein were detected by Coomassie blue stain on SDS-PAGE. Major 36kDa protein was eluted for production of antiserum for serological analysis, IEF and two dimensional electrophoresis. Isoelectric point of 36kDa was aout pH 8.5. Two dimensional electrophoresis of eluted 36kDa showed one point on the gel. Anti-36 kDa serum made by newzilland rabbit for serological test. In ELISA, final titer of antibody was 100×{TEX}$2^5}${/TEX} : 1. Neutralize ability of serum was examined by slide agglutination test and colonization test in rat. Anti-36 kDa serum agglutinated whole cell of V. vulnificus were inhibited colonization on intestine in rat. Accordingly In this paper contain some electrophoretical analysis and serological test of a 36 kDa OMP of V. vulnificus.

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Characterization of the Outer Membrane-Associated 2-Furaldehyde Dehydrogenase from Klebsiella pneumoniae (Klebsiella pneumoniae 균주의 세포외막에서 분리한 2-furaldehyde dehydrogenase의 특성에 관한 연구)

  • 이준우;강사욱;하영칠;한홍의
    • Korean Journal of Microbiology
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    • v.26 no.3
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    • pp.197-206
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    • 1988
  • An outer membrane-associated 2-furaldehyde dehydrogenase, catalyzing the oxidation of 2-furaldehyde to 2-furoic acid from Klebsiella pneumoniae was purified to homogeneity and characterized. The enzyme showed its highly specific dependency on $\beta$-$NAD^{+}$. Enzyme activity was monitored during purification by using substrate 2-furaldehyde and coenzyme $\beta$-$NAD^{+}$ by means of high performance liquid chromatography. The outer membrane was successfully collected by the methods of Percoll density gradient ultracentrifugation and ultracentrifugation after preferential solubilization of the membrane with $Mg^{2+}$ and Triton X-100. The enzyme was purified by the series of procedures including extraction of outer membrane protein with EDTA and lysozume, and fractionation by column chromatography on QAE-Sephades Q-50, and subsequently Sephadex G-100. The enzume showed its optimal activity at $85^{\circ}C$, pH 9.5, and in the presence of 1.5% (vol/vol) Triton X-100. The enzyme exhibited a native molecular size of 88,000 by nondenaturing polyacrylamide gel electrophoresis and had an apparent Km of 4.72mM for 2-furaldehyde.

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