[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes we...[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.展开更多
为探究外膜蛋白A(outer membrane protein A,OmpA)对米尔伊丽莎白菌致病作用的影响,以蛙源米尔伊丽莎白菌FL160902为研究对象,通过同源重组法构建OmpA缺失株△ompA,比较缺失株和野生株的生长特性、生物膜形成能力、抗血清杀伤能力、对...为探究外膜蛋白A(outer membrane protein A,OmpA)对米尔伊丽莎白菌致病作用的影响,以蛙源米尔伊丽莎白菌FL160902为研究对象,通过同源重组法构建OmpA缺失株△ompA,比较缺失株和野生株的生长特性、生物膜形成能力、抗血清杀伤能力、对细胞的黏附能力以及对蛙的致病性差异。结果显示:△ompA的生长能力和抗血清杀伤能力与野生株无显著差异;但与野生株相比,△ompA的生物膜形成能力增加了66%,△ompA对bEnd.3细胞的黏附能力降低了61%;黑斑蛙感染试验显示,△ompA在黑斑蛙血液、脾和脑组织中的载菌量分别为(3.15×10^(8)±0.09×10^(8))、(2.11×10^(8)±0.07×10^(8))和(6.61×10^(8)±0.16×10^(8))copies/g,均显著低于野生株,且△ompA对黑斑蛙的致死率为37%,显著低于野生株的致死率(75%)。上述结果表明,ompA基因缺失不改变米尔伊丽莎白菌的抗血清杀伤能力,但增加了菌株的生物膜形成能力,减弱了菌株的黏附能力,从而降低了该菌对蛙的致病性。展开更多
Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into...Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.展开更多
Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major oute...Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.展开更多
Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolat...Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.展开更多
Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutin...Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.展开更多
Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermo...Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.展开更多
Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchis...Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.展开更多
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer m...Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.展开更多
Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It in...Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.展开更多
AIM: To study immunogenicity of outer membrane protein F(Opr F) fused with B subunit of LT(LTB), against Pseudomonas aeruginosa(P. aeruginosa). METHODS: The Opr F, a major surface exposed outer membrane protein that i...AIM: To study immunogenicity of outer membrane protein F(Opr F) fused with B subunit of LT(LTB), against Pseudomonas aeruginosa(P. aeruginosa). METHODS: The Opr F, a major surface exposed outer membrane protein that is antigenically conserved in various strains of P. aeruginosa, is a promising immunogen against P. aeruginosa. In the present study recombinant Opr F and Opr F-LTB fusion gene was cloned, expressed and purified. BALB/c mice and rabbits were immunized using recombinant Opr F and Opr F-LTB and challenged at the burn site with P. aeruginosa lethal dose of 104 CFU. The protective efficacy of rabbit anti Opr F Ig G against P. aeruginosa burn infection was investigated by passive immunization. RESULTS: It has been well established that the LTB is a powerful immunomodulator with strong adjuvant activity. LTB as a bacterial adjuvant enhanced immunogenicity of Opr F and anti Opr F Ig G titer in serum was increased. Experimental findings showed significantly higher average survival rate in burned mice immunized with Opr F-LTB than immunized with Opr F or the control group. Rabbits anti Opr F Ig G brought about 75% survival of mice following challenge with P. aeruginosa. Post challenge hepatic and splenic tissues of mice group immunized with Opr F-LTB had significantly lower bacterial load than those immunized with Opr F or the control groups. CONCLUSION: These results demonstrate that LTBfused Opr F might be a potential candidate protein for a prophylactic measure against P. aeruginosa in burn infection.展开更多
The correlation between the activities of the outer menbrane proteins （OMPs） of Bordetella pertussis and the lgE-mediated asthma was investigated in the present study, in which the OMPs of B. pertussis and their com...The correlation between the activities of the outer menbrane proteins （OMPs） of Bordetella pertussis and the lgE-mediated asthma was investigated in the present study, in which the OMPs of B. pertussis and their components were prepared by detergent treatment and chromatography, and the molecular weights of the OMPs components were determined by SDS-PAGE. The amounts of total as well as the ovalbumin （OVA）-specific IgE induced by dead B. pertussis whole bacterial vaccine on guinea pigs were detected by ELISA. Meanwhile, the effect of the OMPs and their components to promote the degranulation of guinea pig mast cells was observed by using the mast cell degranulation test, and ELISA assay was used to measure the histatmine levels in the supematants from the mast cell cultures. Histamine sensitive test was used to demonstrate the effects of the OMPs and their components to increase the histamine lethal sensitivity in mice. It was found that four components with molecular weights of 30, 32, 38 and 69 kDa could be obtained from the OMPs of B. pertussts, and the dead whole bacteria vaccine of B. pertussis had the ability to increase the levels of the total as well as the OVA-specific IgE in sera of guinea pigs. The OMPs and their 30 and 32 kDa components demonstrated significantly enhancing effect on the degranulation of guinea pig mast cells, and the histamine levels in the supematants from the mast cell culture treated with OMPs and their 30 and 32 kDa components were also significantly increased. It is evident that the strong adjuvant activity and the enhancing effect to degranulation of mast cells and the release of histamine of certain outer membrane components of B. pertussis could be demonstrated as revealed by the results of the present study, suggesting the possibility of a close relationship between the infection of vaccination with B. pertussis and the IgE-mediated asthma.展开更多
Objective:To investigate the banding patterns of whole cell protein(WCP) and outer membrane protein (OMP) of Burkholderia pseudomallei(B.pseudomallei) in clinical isolates from patients with melioidosis. Methods:WCP a...Objective:To investigate the banding patterns of whole cell protein(WCP) and outer membrane protein (OMP) of Burkholderia pseudomallei(B.pseudomallei) in clinical isolates from patients with melioidosis. Methods:WCP and OMP of of B.pseudomallei in 50 clinical isolates,from 47 patients with melioidosis were prepared and separated by polyacrylamide gel electrophoresis(SDS-PAGE) using 10%gels and stained with Coomassie brilliant blue.The banding patterns were compared by using a laser densitometer and dendrogram. Results:There were 6 different banding patterns of WCP and 2 types of OMP.Type 1 -5 WCP had 8 common protein bands at 19.0 - 45.0 kDa with identical OMP pattern.The banding patterns of WCP in type 6 were distinct from the others and also its OMP profile.The majority of clinical isolates(37/50,74%) were in type 1 WCP.Of the remaining isolates,8 were in type 2,2 in type 3,and one each was in type 4 to 6.There was no significant association between the WCP typing and the demographic or clinical features of the investigated patients.Conclusion:Despite the wide variation of clinical features of melioidosis,the results of this study show that B.pseudomallei had a few differences in the WCP and OMP profiles.Therefore typing of WCP and OMP,using SDS-PAGE analysis,could be an alternative method for phenotypic differentiation in clinical isolates of B.pseudomallei.展开更多
Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic...Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitico. Methods We used SDS-PAGE and western blotting to characterize lipopolysaccharide （LPS）, Yersinio outer membrane proteins （Yops）, membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitico bio-serotype 2/0：9, isolated from China, and highly pathogenic bio-serotype 1B/O：8, isolated from Japan. Results These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. Conclusion The major antigens of the two strains e and membrane proteins, as shown by comparing preparations. citing the host immune protein samples with response were the LPS reference and purified展开更多
基金Supported by China Postdoctoral Science Foundation(20100470565)Science and Technology Support Program of Hebei Province(10960408D)+1 种基金Project of Science and technology Bureau of Shijiazhuang(1150093A)Science and Technology Development Project of Qinhuangdao City(201101A182)~~
文摘[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.
文摘为探究外膜蛋白A(outer membrane protein A,OmpA)对米尔伊丽莎白菌致病作用的影响,以蛙源米尔伊丽莎白菌FL160902为研究对象,通过同源重组法构建OmpA缺失株△ompA,比较缺失株和野生株的生长特性、生物膜形成能力、抗血清杀伤能力、对细胞的黏附能力以及对蛙的致病性差异。结果显示:△ompA的生长能力和抗血清杀伤能力与野生株无显著差异;但与野生株相比,△ompA的生物膜形成能力增加了66%,△ompA对bEnd.3细胞的黏附能力降低了61%;黑斑蛙感染试验显示,△ompA在黑斑蛙血液、脾和脑组织中的载菌量分别为(3.15×10^(8)±0.09×10^(8))、(2.11×10^(8)±0.07×10^(8))和(6.61×10^(8)±0.16×10^(8))copies/g,均显著低于野生株,且△ompA对黑斑蛙的致死率为37%,显著低于野生株的致死率(75%)。上述结果表明,ompA基因缺失不改变米尔伊丽莎白菌的抗血清杀伤能力,但增加了菌株的生物膜形成能力,减弱了菌株的黏附能力,从而降低了该菌对蛙的致病性。
基金This work was supported in part by grants from the Department of Science and Technology of Hunan Province (No. 01SSY2008-6) the Department of Health of Hunan Province (No. B2003-078).
文摘Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.
基金supported by grants from the National Natural Science Foundation of China (Grant No. 30901352)Innovative Research Team in University of Hunan Province (Number: [2008] 51)Hunan Provincial Innovation Foundation for Postgraduate and Hunan Provincial Training and Innovation Base for Post-graduate
文摘Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.
基金the National Natural Science Foundation of China(31072151)the Specialized Research Fund for the Doctoral Program of Higher Education,China(20090097110007)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)
文摘Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.
文摘Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.
基金Supported by the National Basic Research Program of China(973 Program)(No.2009CB118703)the Science and Technology Program of Xiamen Southern Oceanographic Center(No.14PYY050SF03)the National Science and Technology Support Program Project of China(No.2012BAD25B02)
文摘Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.
基金supported by the China Agricultural Research System(nycytx-44-3-2)the Zhejiang Key Project on Agricultural Development through Science and Technology(2011C12028)+1 种基金the National Natural Science Foundation of China(31302068)the Zhejiang Provincial Natural Science Foundation of China(LQ13C180002)
文摘Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.
基金Supported by the Key Laboratory Foundation of the Educational Department of Liaoning Province (No. 2009S024)the Dalian Municipal Government of China (No. 2007B11NC069)the Grant of Dalian Ocean University (No. SY2007005)
文摘Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.
基金Supported by the Research Plan of Jiangsu Provincial Technology Commission, No. BS2004021Advanced Talent Research Plan of Jiangsu University, No. JDG2004008
文摘Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.
文摘AIM: To study immunogenicity of outer membrane protein F(Opr F) fused with B subunit of LT(LTB), against Pseudomonas aeruginosa(P. aeruginosa). METHODS: The Opr F, a major surface exposed outer membrane protein that is antigenically conserved in various strains of P. aeruginosa, is a promising immunogen against P. aeruginosa. In the present study recombinant Opr F and Opr F-LTB fusion gene was cloned, expressed and purified. BALB/c mice and rabbits were immunized using recombinant Opr F and Opr F-LTB and challenged at the burn site with P. aeruginosa lethal dose of 104 CFU. The protective efficacy of rabbit anti Opr F Ig G against P. aeruginosa burn infection was investigated by passive immunization. RESULTS: It has been well established that the LTB is a powerful immunomodulator with strong adjuvant activity. LTB as a bacterial adjuvant enhanced immunogenicity of Opr F and anti Opr F Ig G titer in serum was increased. Experimental findings showed significantly higher average survival rate in burned mice immunized with Opr F-LTB than immunized with Opr F or the control group. Rabbits anti Opr F Ig G brought about 75% survival of mice following challenge with P. aeruginosa. Post challenge hepatic and splenic tissues of mice group immunized with Opr F-LTB had significantly lower bacterial load than those immunized with Opr F or the control groups. CONCLUSION: These results demonstrate that LTBfused Opr F might be a potential candidate protein for a prophylactic measure against P. aeruginosa in burn infection.
文摘The correlation between the activities of the outer menbrane proteins （OMPs） of Bordetella pertussis and the lgE-mediated asthma was investigated in the present study, in which the OMPs of B. pertussis and their components were prepared by detergent treatment and chromatography, and the molecular weights of the OMPs components were determined by SDS-PAGE. The amounts of total as well as the ovalbumin （OVA）-specific IgE induced by dead B. pertussis whole bacterial vaccine on guinea pigs were detected by ELISA. Meanwhile, the effect of the OMPs and their components to promote the degranulation of guinea pig mast cells was observed by using the mast cell degranulation test, and ELISA assay was used to measure the histatmine levels in the supematants from the mast cell cultures. Histamine sensitive test was used to demonstrate the effects of the OMPs and their components to increase the histamine lethal sensitivity in mice. It was found that four components with molecular weights of 30, 32, 38 and 69 kDa could be obtained from the OMPs of B. pertussts, and the dead whole bacteria vaccine of B. pertussis had the ability to increase the levels of the total as well as the OVA-specific IgE in sera of guinea pigs. The OMPs and their 30 and 32 kDa components demonstrated significantly enhancing effect on the degranulation of guinea pig mast cells, and the histamine levels in the supematants from the mast cell culture treated with OMPs and their 30 and 32 kDa components were also significantly increased. It is evident that the strong adjuvant activity and the enhancing effect to degranulation of mast cells and the release of histamine of certain outer membrane components of B. pertussis could be demonstrated as revealed by the results of the present study, suggesting the possibility of a close relationship between the infection of vaccination with B. pertussis and the IgE-mediated asthma.
基金supported by the Mahidol-Oxford Tropical Medicine Research Unit,UK.
文摘Objective:To investigate the banding patterns of whole cell protein(WCP) and outer membrane protein (OMP) of Burkholderia pseudomallei(B.pseudomallei) in clinical isolates from patients with melioidosis. Methods:WCP and OMP of of B.pseudomallei in 50 clinical isolates,from 47 patients with melioidosis were prepared and separated by polyacrylamide gel electrophoresis(SDS-PAGE) using 10%gels and stained with Coomassie brilliant blue.The banding patterns were compared by using a laser densitometer and dendrogram. Results:There were 6 different banding patterns of WCP and 2 types of OMP.Type 1 -5 WCP had 8 common protein bands at 19.0 - 45.0 kDa with identical OMP pattern.The banding patterns of WCP in type 6 were distinct from the others and also its OMP profile.The majority of clinical isolates(37/50,74%) were in type 1 WCP.Of the remaining isolates,8 were in type 2,2 in type 3,and one each was in type 4 to 6.There was no significant association between the WCP typing and the demographic or clinical features of the investigated patients.Conclusion:Despite the wide variation of clinical features of melioidosis,the results of this study show that B.pseudomallei had a few differences in the WCP and OMP profiles.Therefore typing of WCP and OMP,using SDS-PAGE analysis,could be an alternative method for phenotypic differentiation in clinical isolates of B.pseudomallei.
基金supported by National Natural Science Foundation of China (General Project,No. 30970094)National Sci-Tech Key Project (2009ZX10004-201,2009ZX10004-203)
文摘Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitico. Methods We used SDS-PAGE and western blotting to characterize lipopolysaccharide （LPS）, Yersinio outer membrane proteins （Yops）, membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitico bio-serotype 2/0：9, isolated from China, and highly pathogenic bio-serotype 1B/O：8, isolated from Japan. Results These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. Conclusion The major antigens of the two strains e and membrane proteins, as shown by comparing preparations. citing the host immune protein samples with response were the LPS reference and purified
