Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

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.

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

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.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

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.

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

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.

30 and 32 kDa outer membrane proteins of Bordetella pertussis as a modulator on promoting degranulation of mast cells

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.

Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[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.

Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

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.

Development and Evaluation of a MAb-Based ELISA for Detection of Chlamy- dophila pneumoniae Infection with Variable Domain 2 and 3 of the Major Outer Membrane protein

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.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

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.

Research progress on Helicobacter pyloriouter membrane protein

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.

Recombinant outer membrane protein F-B subunit of LT protein as a prophylactic measure against Pseudomonas aeruginosa burn infection in mice

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.

军团菌MOMPS基因的克隆并在原核系统中表达

以军团菌DNA为模板,PCR扩增获得军团菌主要外膜蛋白基因(M a jor ou ter m em brane prote in gene,om pS),与原核表达质粒pUC 18定向重组,构建重组质粒,转化大肠杆菌BL 21,并用限制性酶酶切分析、聚合酶链式反应、核酸序列分析、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、W estern印迹进行鉴定。实验结果表明我们扩增出了军团菌914 bp的om pS基因,成功构建了重组质粒pLPom pS,并在原核系统中得到了表达。

HopQ-CEACAM复合体在 H.pylori 阳性胃癌中的研究进展

胃癌严重影响人类健康,是全球最常见的恶性肿瘤之一。幽门螺杆菌(Helicobacterpylori,H.pylori)是人胃上皮细胞的特异性定植菌,是引起胃炎、消化性溃疡以及胃癌的重要原因之一。H.pylori凭借其外膜蛋白家族的成员H.pylori外膜蛋白Q(H.pylori outer membrane protein,HopQ)可以与人体宿主细胞表面的人癌胚抗原相关细胞黏附分子1(carcinoembryonic antigen-related cell adhesion molecule 1,CEACAM1)结合,形成Hop-CEACAM1复合体,将细胞毒素相关基因A(cytotoxin-associated gene A,CagA)注入到宿主细胞,发挥毒性作用,产生慢性炎症,若未进行治疗就会导致癌症的发生。本文就Hop-CEACAM1复合体的组成及相互作用以及在H.pylori阳性胃癌中的机制进行综述。

SDS-PAGE analysis of whole cell protein and outer memrbane protein patterns of clinical isolates of Burkholderia pseudomallei

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.

Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins(OMPs)of Flavobacterium columnare

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.

Genotypic Analysis, Construction of the Expression System and Immunological Identification of the Recombinant Proteins of the LipL32 Gene in the Dominant Serogroups of Leptospira interrogans in China

To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.

鲍曼不动杆菌OmpA蛋白的生物学特性分析

目的提取临床分离的鲍曼不动杆菌外膜蛋白A(OmpA)的基因,并进行生物学预测和分析。方法查询鲍曼不动杆菌OmpA基因序列,设计特异性PCR引物,以提取的鲍曼不动杆菌基因组DNA为模板,PCR扩增OmpA片段。回收目的片段,采用生物信息学软件分析OmpA蛋白的生物学特性。结果12株鲍曼不动杆菌多位点序列分型(MLST)序列分析有6个ST分型,分别是ST 208、ST 229、ST 191、ST 195、ST540、ST 1145,鲍曼不动杆菌OmpA基因序列全长为1070 bp的,单核苷酸多态性(SNP)位点较少,预测蛋白为跨膜蛋白,具有6种三级结构,均由β-桶状结构组成的保守结构域。结论临床分离的不同鲍曼不动杆菌株OmpA蛋白为结构相对保守的跨膜蛋白,具有维持细胞的形状和稳定性,参与细菌耐药机制及免疫原性作用。

产金属β-内酰胺酶大肠埃希菌对亚胺培南的耐药性研究

目的:分析产金属β-内酰胺酶大肠埃希菌对亚胺培南和美罗培南敏感性降低的原因。方法:用脉冲场凝胶电泳(PFGE)进行同源性分析,采用E-test方法测定菌株及接合子和转化子的药敏情况。采用RT-PCR扩增和序列分析、碳青霉烯酶抑制剂增强实验、外膜蛋白分析(SDS-PAGE)以及羰酰氰氯苯腙(CCCP)抑制试验分析大肠埃希菌对亚胺培南耐药的原因。结果:菌株Eco1、Eco2和Eco3为非同一克隆株,对亚胺培南的MIC分别为6μg/mL、16μg/mL和16μg/mL,对美罗培南的MIC分别为6μg/mL、16μg/mL和32μg/mL,对厄他培南、哌拉西林/他唑巴坦耐药、氨曲南、头孢噻肟、头孢曲松、头孢吡肟、头孢西丁、左氧氟沙星和庆大霉素均高度耐药;菌株Eco3对阿米卡星敏感,菌株Eco1和Eco2对阿米卡星高度耐药。菌株Eco1产CTX-M-79、SHV-11、IMP-4、CMY-6和NDM-1,菌株Eco2产CTX-M-79、CTX-M-14、CMY-6和NDM-1,菌株Eco3产CTX-M-14、CMY-6和IMP-4。CCCP不能提高Eco1、Eco2和Eco3对亚胺培南的敏感性,SDS-PAGE发现菌株Ompk35和Ompk36未见缺少,3株大肠埃希菌EDTA增强实验阳性。仅菌株Eco1的NDM-1金属β-内酰胺酶通过接合试验传递到大肠埃希菌J53上,其接合子对亚胺培南和美罗培南耐药,EDTA增强实验阳性;菌株Eco2接合子和转化子不含NDM-1金属β-内酰胺酶,其接合子和转化子对亚胺培南和美罗培南敏感;菌株Eco3接合试验和转化试验未成功。结论:同时产CTX-M-79、CTX-M-14、CMY-6、NDM-1、IMP-4或CTX-M-79、CTX-M-14、CMY-6、IMP-4β-内酰胺酶的大肠埃希菌可引起亚胺培南高度耐药。

基于外膜蛋白的鼻气管鸟杆菌抗体间接ELISA检测方法的建立

【目的】建立鼻气管鸟杆菌(Ornithobacterium rhinotracheale,ORT)血清抗体的ELISA检测方法。【方法】利用原核表达方法克隆表达鼻气管鸟杆菌外膜蛋白OR02,经Western blotting鉴定其免疫原性。以纯化产物作为抗原包被于固相载体,采用棋盘滴定法确定抗原包被浓度和血清稀释度。同时,优化ELISA反应条件,利用鼻气管鸟杆菌阴性血清确定该检测方法的阴阳临界值判定标准。通过检测其他病原阳性血清评估该方法的特异性,检测不同比例稀释的鼻气管鸟杆菌阳性血清以评估其灵敏性,并与商品化试剂盒进行比较,评估该ELISA方法的符合率。【结果】重组蛋白OR02以包涵体形式表达,分子质量约为58 ku,与预期相符。Western blotting显示,OR02重组蛋白能与鼻气管鸟杆菌阳性血清发生特异性结合,具有良好的免疫反应性。以纯化的OR02蛋白作为包被抗原,确定最佳抗原包被浓度为2 ng/μL,血清样本的稀释度为1∶50,孵育时间为30 min,酶标二抗的稀释度为1∶5000,孵育时间为30 min,最佳底物显色条件为在避光条件下孵育15 min。阴阳临界值的判定标准为:当待检血清D_(450 nm)值≥0.266时判定为阳性,当待检血清D_(450 nm)值<0.266时判定为阴性。用该方法检测副鸡禽杆菌(Apg)、新城疫病毒(NDV)、鸡传染性法氏囊病毒(IBDV)等单因子阳性鸡血清均无交叉反应。将抗体滴度为1∶64的鼻气管鸟杆菌阳性鸡血清以1∶800稀释仍能检测到阳性结果。与商业试剂盒对相同临床样本的检测结果的符合率为91.25%。【结论】本研究利用重组OR02蛋白建立了一种检测鼻气管鸟杆菌血清抗体的间接ELISA方法,该方法具有良好的特异性和灵敏性,且与商业试剂盒的符合率较高,可作为鸡血清鼻气管鸟杆菌抗体的检测工具。

基于不同抗原的犬布鲁氏菌病免疫层析胶体金抗体试纸条的制备和比较

为建立针对犬布鲁氏菌病的免疫层析胶体金快速检测方法,利用从犬种布鲁氏菌菌株提纯的膜蛋白和可溶性蛋白,原核表达纯化的外膜蛋白Omp31和BP26以及基于B细胞抗原肽的融合蛋白F14,分别制备了试纸条,然后采用犬布鲁氏菌病阳性和阴性血清,分别对上述试纸条进行敏感性和特异性比较。结果显示:Omp31抗原的敏感性最高,其次是F14和BP26,而全菌膜蛋白和全菌可溶性蛋白的敏感性较差;特异性上,使用由5种抗原制备的胶体金试纸条检测30份犬布鲁氏菌病阴性血清,结果均为阴性;5种试纸条与沙门氏菌、大肠杆菌(O157)、大肠杆菌(H7)、霍乱弧菌、副溶血弧菌、军团菌阳性血清均不发生交叉反应。另外,由F14和BP26制备的试纸条可识别羊布鲁氏菌病阳性血清,而由Omp31、全菌膜蛋白和全菌可溶性蛋白制备的试纸条均不能识别牛、羊布鲁氏菌病阳性血清。综上所述,Omp31、F14和BP26可作为制备犬布鲁氏菌病免疫层析胶体金检测卡的备选抗原。本研究为粗糙型布鲁氏菌引起的布鲁氏菌病的诊断提供了技术支撑。