布鲁氏菌重要膜蛋白研究进展

布病是由布鲁氏菌属细菌引起的一种重要的人兽共患病,在世界范围内广泛分布。布病不仅可导致巨大的经济损失,也是威胁人群健康的主要风险因素。布鲁氏菌外膜蛋白是主要的免疫和保护性抗原,不仅与布鲁氏菌的毒力和细胞内生存有密切的关联,而且对开发和建立新型的特异性血清学诊断方法具有重要的意义。本文对布鲁氏菌的重要外膜蛋白的研究进展予以综述,从而更好的理解布鲁氏菌表面蛋白的抗原特性,为建立布病实验室诊断方法和新型疫苗研发提供参考。     

快速免疫诊断牛布鲁氏菌病新方法的研究

目的建立一种牛布鲁氏菌病快速免疫诊断方法。方法斑点金免疫渗滤法(DIGFA)以硝酸纤维素膜为载体,胶体金标记蛋白为显示剂,抗原、抗体及胶体金标记蛋白通过渗滤发生反应,阳性结果在膜上显示红色斑点。结果DIGFA快速检测牛布鲁氏菌病具有很好的重现性和稳定性;敏感性、特异性分别达到97%和100%。结论DIGFA快速检测牛布鲁氏菌病,方法快速简便,测试过程1~2 min内完成,试剂敏感、特异、稳定,非常适合于布鲁氏菌病的诊断及流行病学调查。     

不同种布鲁氏菌膜蛋白抗原与布鲁氏菌感染豚鼠血清的ELISA试验

<正> 有关布鲁氏菌膜蛋白(OMP)的研究,近几年引起国内外学者的关注,国内武素怀(1991)曾报道有关外膜蛋白的电泳图谱特点,但采用布鲁氏菌OMP为抗原检测相应抗体的报道并不多见。针对上述情况,我们用S型及R型布鲁氏菌OMP为抗原,检测不同种布鲁氏菌感染动物的抗体反应,同时观察其用于鉴别Y·e O:9感染的可能性。     

羊种布鲁氏菌抗原成分分析及其保护性鉴定

分析了羊种布鲁氏菌 M28、M5、M5—90三菌株的胞浆蛋白、菌壁、外膜蛋白及脂多糖四种抗原成分及其氨基酸组成,比较了强毒菌株 M28与弱毒菌株 M5和 M5—90以及两菌株之间的异同,并测得四种抗原成分的保护率分别为菌壁100%=脂多糖100%>外膜蛋白75%>胞浆蛋白40%,以及各抗原成分引起血清中凝集抗体的出现情况。     

建立双抗原夹心酶免疫试验对人畜布鲁氏菌抗体检测的研究

目的为人畜布鲁氏菌病(布病)的血清学诊断、监测及流行病学调查提供特异、敏感、快速的试验方法。方法在制备高滴度的布鲁氏菌(布氏菌)抗原及其抗原辣根过氧化物酶结合物基础上。建立了检测人畜布氏菌抗体的双抗原夹心酶免疫试验(DAgS-EIA),包括常规双抗原夹心酶联免疫吸附试验(DAgS-ELISA)、双抗原夹心酶免疫斑点试验(DAgS-DIEA)以及快速双抗原夹心ELISA(快速DAgS-ELISA),并对影响本试验的主要因素因素进行了试验。结果制备了高滴度的布氏菌抗原及其抗原酶结合物和冻干制品。并在此基础上,建立了常规DAgS-ELISA及DAgS-DIEA以及快速DAgS-ELISA检测人畜布氏菌抗体方法。经对115份布病患者血清检测结果,阳性率以DAgS-ELISA为最高(61.7%),其余依次为RBPT(58.1%)I、-ELISA(55.6%)、DAgS-DIEA(53.7%)、及SAT(44.2%)且用DAgS-EIA检测布氏菌感染羊、牛、猪及实验动物家兔、豚鼠和小鼠均为强阳性反应,而对正常人、羊、牛、猪及实验动物血清均为阴性反应。结论双抗原夹心酶免疫试验检测人畜血清布氏菌抗体,不仅特异、敏感、快速、简便而且仅需制备单一的布氏菌抗原酶结合物就可对人及各类动物血清布氏菌抗体进行检测。     

布鲁氏菌BP26蛋白的表达纯化及其抗原性的研究

目的获得布鲁氏菌感染血清学诊断优势抗原BP26重组蛋白。方法应用PCR技术从布鲁氏菌减毒株(104M)中扩增出bp26目的基因片段,将其克隆于表达载体PET30a中,并转化入大肠杆菌BL21(DE3)中,IPTG诱导表达,用亲和层析纯化BP26重组蛋白,然后分别用Western-blot,间接ELISA检测其抗原性。结果DNA测序及酶切鉴定证实PET-30a/bp26原核表达载体构建成功,并在大肠杆菌中高效表达,免疫印迹和间接ELISA实验证明BP26重组蛋白可与布鲁氏菌阳性血清产生特异性结合。结论成功获得了布鲁氏菌中序列全长为696bp,编码232个氨基酸的BP26蛋白,通过血清学反应证实,该蛋白为人和动物布鲁氏菌病临床诊断试剂盒的研发奠定了基础。     

羊布鲁氏菌BCSP31、OMP31、L7/L12基因的原核表达载体构建及表达

目的构建羊布鲁氏菌外膜蛋白BCSP31、OMP31和L7/L12基因的重组表达质粒,并在原核系统中表达。方法根据羊布鲁氏菌M5株外膜蛋白BCSP31、OMP31和抗原L7/L12蛋白基因序列设计引物,分别扩增出大小约为1kb、730bp和380bp的目的基因片断,插入pGEM7Zf(+)中测序并进行分析。将3种基因片段亚克隆入pGEX-4T-1中,转化大肠杆菌,IPTG诱导表达,用SDS-PAGE及Western-blot进行分析鉴定。结果SDS-PAGE结果表明3个目的蛋白均以融合蛋白的方式成功表达,在相对分子量57kDa、49kDa、38kDa处有表达条带,经薄层扫描分析,目的蛋白分别占全菌蛋白的42%、36%、40%。Western-blot证实3种融合蛋白均能被布鲁氏菌免疫兔血清识别。结论成功构建了3种蛋白的表达载体并进行了高效表达,3种蛋白均具有良好的免疫原性。     

布鲁氏菌BsAg抗原的免疫电镜观察

利用免疫电镜包埋前包埋后染色法，分别观察６种布鲁氏菌及耶尔森氏０：９型菌中ＢｓＡｇ的分布情况，发现ＢｓＡｇ主要分布在布鲁氏菌外膜，菌体细胞质内很少，以１６Ｍ羊种布鲁氏菌株中ＢｓＡｇ抗原最多，其余５种布鲁氏菌株中ＢｓＡｇ较少。用包埋前染色法未发现耶尔森氏０：９型菌外膜表面有ＢｓＡｇ存在，而用包埋后染色法观察到布鲁氏菌体细胞质内及耶尔森氏０：９型菌外膜内侧和菌体细胞质内有微量的ＢｓＡｇ。提示布鲁氏菌细     

布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立

目的建立一种快速检测布鲁氏菌抗体的新方法。方法利用胶体金免疫层析技术,以大肠埃希菌表达、纯化的OMP31与BP26重组蛋白作为检测抗原,以金黄葡萄球菌A蛋白(SPA)作为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁氏菌抗体,并分析方法的敏感性和特异性。结果以粒径为40nm胶体金制备的试纸条检测布鲁氏菌抗体的敏感性最高,胶体金最佳标记pH为6.2,SPA蛋白最适标记量为6μg/ml。交叉试验证明试纸条不与其他非相关疾病感染血清反应,特异性高。试纸条检测结果与琥红平板试验方法的符合率为92%。结论制备的布鲁氏菌抗体胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,可用于鉴别布鲁氏菌自然感染和人工免疫,并可区分牛、羊种布鲁氏菌感染,可在基层推广使用。     

用协同凝集试验检测布鲁氏菌

用葡萄球菌甲蛋白(SPA)协同凝集试验检测细菌抗原具有敏感而特异等优点,我们用此试验检测布鲁氏菌,并比较了检测活菌与死菌颗粒性抗原同超声波粉碎可溶性抗原,以及影响试验的因素等结果。 试验方法:用布氏菌104M株免疫家兔制备抗布氏菌的免疫血清,用DEAE纤维素批量法提取IgG,再用金黄色葡萄球菌1800株制备10％的SPA稳定液,将抗血清和提取的IgG分别致敏SPA稳定液,取此稳定液一     

布鲁氏菌bp26和OMP10基因的原核表达和鉴定

目的研究布鲁氏菌外膜蛋白OMP10和bp26基因的表达、克隆与测序,并对其进行初步的血清学鉴定。方法从布鲁氏菌基因组中获得OMP10和bp26基因连接入PMD18-T克隆质粒并测序,克隆入融合表达载体PGEX-4T-1,构建重组质粒PGEX-4T-1/OMP10和PGEX-4T-1/bp26,在大肠杆菌中将该蛋白表达。用western-blot分析重组表达的GST-OMP10和GST-bp26的免疫学特性。结果成功构建了PGEX-4T-1/OMP10和PGEX-4T-1/bp26原核表达载体,并在大肠杆菌中成功表达了OMP10和bp26基因,布鲁氏菌免疫动物血清能特异性识别所表达的蛋白。结论布鲁氏菌外膜蛋白OMP10和bp26基因的成功表达,它可以与布鲁氏菌免疫血清产生特异性结合反应,为之后的抗原性以及免疫原性研究打下良好的物质基础。     

Serologic diagnosis of pertussis: evaluation of pertussis toxin and other antigens in enzyme-linked immunosorbent assay

IgM, IgA, and IgG antibodies to Bordetella pertussis were measured in paired sera from 34 patients who were culture-positive for pertussis by enzyme-linked immunosorbent assay (ELISA) with disrupted B. pertussis bacteria, purified pertussis toxin, or outer membrane proteins (OMP) as antigens. Paired sera from 50 patients with other respiratory infections were used as controls. The sensitivities of the assays from paired sera were 61%, 90%, and 90% and specificities were 98%, 92%, and 72%, respectively. Of the patients culture-positive for pertussis, 68% had positive levels of antibody to pertussis toxin antigen in their first serum samples, obtained at the same time as samples for culture. Infants had antibody responses to pertussis toxin antigen, in contrast to weak antibody responses measured by B. pertussis antigen. The results from this study indicate that ELISA, especially measuring pertussis toxin IgA, is a valuable additional tool for diagnosing pertussis and can be used as a complementary test with cultures.     

Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus

For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.     

犬种布鲁氏菌外膜蛋白的特征及宿主牦牛对其外膜蛋白的影响

SDS—PAGE揭示出犬种布鲁氏菌外膜蛋白具有布鲁氏菌属的共同性,同时也具有其种的特征性。其独特表现为:具有一组非热修饰蛋白成分;且其微孔外膜蛋白解聚后除解聚成正常单体外,还有一组特殊的热修饰组分,此二成分受不同宿主影响而有所差异。     

弓形虫速殖子表膜蛋白的组分和抗原性分析

目的试验分析弓形虫速殖子表膜蛋白的组分及其抗原性。方法用聚丙烯酰胺凝胶电泳分析弓形虫速殖子表膜蛋白的组分和用弓形虫病人阳性血清及兔抗弓形虫表膜抗原血清识别硝酸纤维膜载体上的速殖子表膜抗原的免疫反应靶位。结果弓形虫速殖子表膜抗原的独特蛋白区带分子为16和17kDa,弓形虫病人特异性抗血清识别的速殖子表膜抗原的免疫反应位点为16kDa,免抗血清识别出64,35,32,26,16kDa等8个位点。结论弓形虫速殖子表膜蛋白存在特异的抗原决定簇和可被特异性抗体识别的受体,证明速殖子表膜蛋白具有较好的抗原性,此对于弓形虫病的免疫学诊断具有重要的意义。     

Reactivities and clinical relevance of antimitochondrial antibodies to four mitochondrial inner membrane proteins in sera of patients with primary biliary cirrhosis

Antimitochondrial antibodies are characteristically detected in sera of patients with primary biliary cirrhosis. The antigens to which the antimitochondrial antibodies in primary biliary cirrhosis sera react have been located in the mitochondrial inner membrane. We have reported on four mitochondrial inner membrane proteins, extracted from beef heart, which reacted with antimitochondrial antibodies of primary biliary cirrhosis. These four proteins had molecular weights of 70, 54, 51 and 45 kd. Forty-six of the 50 PBC sera tested were positive with rat kidney and stomach specimens by the indirect immunofluorescent method. All sera of 50 primary biliary cirrhosis patients were positive with at least one and as many as four of the mitochondrial proteins extracted from beef heart using immunoblotting. Forty-seven primary biliary cirrhosis sera (94%) had IgG antibodies (antimitochondrial antibodies) to the antigen proteins, 43 patients (86%) IgM antimitochondrial antibodies and 38 patients (76%) IgA antimitochondrial antibodies. Seventy per cent of patients had antimitochondrial antibodies in all three immunoglobulin classes. In a few primary biliary cirrhosis sera, antimitochondrial antibodies were represented by only one class of immunoglobulin. The major antigenic proteins of mitochondrial inner membrane to which the antimitochondrial antibodies react were the 70 and 51 kd proteins. All primary biliary cirrhosis sera containing antibodies to the 54 and/or 45 kd proteins reacted with 70 kd as well. Patients with an elevation of serum bilirubin over 2 mg per dl were often found to produce antimitochondrial antibodies reactive with the 54 kd in addition to the 70 kd protein. No significant association was found between histological classifications and reactive patterns of antimitochondrial antibodies.     

Use of human chondrocyte cell cultures to identify and characterize reactive antibodies in rheumatoid arthritis sera

OBJECTIVE: To study the reactivity of rheumatoid arthritis (RA) sera with human chondrocyte populations isolated from normal cartilage and expanded in vitro. METHODS: Human articular chondrocytes were cultured as adherent (non-differentiated) cells on plastic dishes or in suspension (differentiated) on dishes previously coated with a thin layer of 1% agarose. Sera from 28 RA patients and 5 paired synovial fluids were tested on lysates from chondrocytes and fibroblasts as control by immunoblot. Antigen expression on the cell membrane was evaluated by flow cytometry in a few sera. RESULTS: In 9/28 RA sera IgG antibodies specific for chondrocyte antigens (97 kDa, 74 kDa, 67 kDa, 60 kDa, 54 kDa, 48 kDa and 37 kDa) were detected. Twelve sera reacted with proteins expressed both on chondrocytes and fibroblasts and 7 with fibroblasts only; two sera had no reactivity. When lysates from adherent or suspension chondrocytes were compared, RA sera reacted with higher intensity and detected more antigens on chondrocytes cultured in suspension. Flow cytometry assay demonstrated that RA sera are able to recognize antigens expressed on the cell membrane of the human chondrocytes. CONCLUSION: Our data indicate that: a) 32% of the RA sera contain antibodies reactive with antigens expressed exclusively by chondrocytes, but this value rises to 75% if antigens expressed both by chondrocytes and fibroblasts are considered; b) the reactivity of fully differentiated chondrocytes in suspension culture is higher than the reactivity of chondrocytes cultured in monolayer; and c) some of the chondrocyte-specific antigens identified are associated with the chondrocyte membrane. Thus, in vitro cultured chondrocytes may be used to study both the specificity and the biological activity of autoantibodies in RA.     

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.     

Patients with active Crohn's disease have elevated serum antibodies to antigens of seven enteric bacterial pathogens

A variety of bacterial pathogens including Campylobacter, Yersinia, Listeria, Brucella, and Mycobacteria have been suggested as potential etiologic agents for Crohn's disease. To assess the role of these organisms we studied responses to eight antigens in sera from patients with active Crohn's disease and healthy age- and sex-matched controls. In complement-fixation assays, the sera from the Crohn's disease patients had enhanced reactivity compared with the control sera to all seven orally ingested pathogens studied; however, only the difference in distribution of titers to Yersinia pseudotuberculosis was statistically significant (p less than 0.0025). There was no difference between the two groups in reactivity to arabinomannan, a common mycobacterial antigen. Seroreactivity to enteric pathogens not resident in the bowel flora probably represents a nonspecific sensitization to cross-reacting antigens. Lack of response to the mycobacterial antigen suggests that widespread mycobacterial disease with high bacillary load is not present in Crohn's disease.     

Identification of platelet proteins that bind alloantibodies and autoantibodies

We have used the techniques of radioimmunoprecipitation (RIP) and Western blot to identify the membrane proteins that bind certain alloantibodies. Anti-PlA1 sera precipitated two bands, corresponding to platelet glycoproteins IIb and III, whether or not calcium was present during the procedure. By Western blot, this antibody bound only glycoprotein III. Anti-PlA1 serum does not precipitate proteins from the platelets of a patient with Glanzmann's thrombasthenia. Two monoclonal antibodies reacting with lymphocyte HLA antigens, as well as sera from highly allosensitized patients, precipitated bands of 38,500 and 13,500 daltons. These bands correspond to the molecular weights of the two subunits of the HLA antigen, as it has been described for other cell types. The patients' sera also precipitated a protein of 72,000 daltons from some platelets. The sera of two patients with quinidine- induced thrombocytopenia precipitated a 138,000-dalton band (glycoprotein Ib-alpha) in the presence of quinidine. The purified IgG antibody from one patient did not require other plasma factors to bind to platelets in the presence of quinidine, while purified antibody from a second patient required plasma factors other than, or in addition to von Willebrand factor. Although several sera from patients with idiopathic thrombocytopenic purpura (ITP) were tested, only one precipitated membrane proteins by the RIP method; this serum identified binding proteins corresponding to glycoproteins IIb and III.