A组β溶血性链球菌脂磷壁酸抗体在风湿热诊断中的意义

目的 探讨A组β溶血性链球菌（乙链菌）脂磷壁酸（LTA）抗体在风湿热诊断中的意义。方法 用从A组乙链菌中提取的LTA作为抗原，采用斑点酶联免疫吸附测定（ELISA）法检测血清中的LTA抗体。结果 ①风湿性心脏炎组LTA－IgG抗体滴度水平高于静止期风湿性心脏病组、病毒性心肌炎组、非湿性心脏瓣膜病组，其他关节炎组的滴度水平；②活动期风湿热患者LTA－IgG抗体阳性率（64％）高于对照组的阳性率；③风湿性心脏炎患者的LTA－IgG抗体阳性率与外周血淋巴细胞促凝血活性（PCA）、抗链球菌壁多糖抗体（ASP）、抗DNAse－B、血沉、C反应蛋白的阳性率相接近，高于抗链球菌溶血素O(ASO)的阳性率。结论 血清LTA抗体在判断风湿热和风湿性心脏病风湿活动有较高的敏感性和一定的特异性。     

亲和层析法纯化华支睾吸虫抗原的研究

目的探索华支睾吸虫特异性抗原的纯化方法。方法从华支睾吸虫病流行区的淡水鱼中分离华支睾吸虫囊蚴,接种动物,收集成虫,制备匀浆液(粗抗原),借助偶联华支睾吸虫病人血清抗体的Sepharose4B亲和层析柱,提取特异性抗原,用浓度梯度聚丙烯酰胺凝胶电泳(CG-PAGE)和免疫印迹方法鉴定纯化抗原的纯度和特异性。结果亲和层析法纯化抗原经CG-PAGE显示5条蛋白带,免疫印迹试验鉴定均有抗原性,强阳性带蛋白分子质量单位为31ku,各区带蛋白与并殖吸虫、血吸虫血清抗体无交叉反应。粗抗原有16条蛋白带。结论经亲和层析法纯化的华支睾吸虫抗原特异性高。亲合层析是获取华支睾吸虫诊断用抗原的较佳方法。     

幽门螺杆菌VacA蛋白的纯化

目的对幽门螺杆菌(Hp)相对分子质量(Mv)95×10~2的VacA蛋白进行分离纯化。为进一步研究VaGA的作用机制奠定基础.方法采用经硫酸铵沉淀及透析后得到的Hp ccUG17874菌株浓缩上清,应用吸附层析、疏水层析、阴离子交换层析及凝胶过滤等液相层析方法进行VacA蛋白的纯化.每步层析后得到的样品均检测蛋白浓度及空泡毒素活性并行SDS-PAGE电泳检测蛋白成分.结果纯化蛋白的Mr为95×10~3,电泳纯,具有明显的空泡毒性,比活力达20480,与上清原液相比纯化倍数提高了4550倍,产率约为5μg/L.结论采用吸附层析、疏水层析、离子交换层析及凝胶过滤的纯化流程,可以成功分离纯化Hp液体培养上清液中的VacA,纯化的VacA蛋白可用于Hp致病机理的研究.     

重组日本血吸虫极低密度脂结合蛋白的抗原性及诊断价值的研究

目的 探讨重组日本血吸虫特异性极低密度脂结合蛋白(SVLBP)的抗原性及其诊断价值。方法 SVLBP基因亚克隆入pQE-30表达载体，克隆菌在IPTG诱导下表达重组蛋白；非变性条件下制备克隆菌裂解液，用亲和层析法纯化重组蛋白；用Western blot鉴定其抗原性；纯化SVLBP重组蛋白免疫家兔获得抗血清；以SVLBP重组蛋白为抗原。ELISA法检测血吸虫感染者血清及健康人血清。结果 克隆菌在IPTG诱导下高表达SVLBP重组蛋白；该重组蛋白诱导家兔产生单特异抗体，抗体滴度为1：6400；SVLBP重组蛋白与血吸虫病人血清有较强的反应；以该蛋白为抗原检测36人份血吸虫感染者血清，阳性率为83．3％，50人份健康人血清，假阳性率为2％(1／50)，10人份肺吸虫病人血清，1例阳性。交叉反应率为10％(1／10)。结论 SVLBP重组蛋白有较好的抗原性，以该重组蛋白为抗原检测日本血吸虫病患者血清中特异性抗体。敏感性和特异性均较高。具有一定的诊断价值。     

亲和层析法纯化华支睾吸虫抗原的研究

目的 探索华支睾吸虫特异性抗原的纯化方法。方法从华支睾吸虫病流行区的淡水鱼中分离华支睾吸虫囊蚴，接种动物，收集成虫，制备匀浆液（粗抗原），借助偶联华支睾吸虫病人血清抗体的Sepharose4B亲和层析柱，提取特异性抗原，用浓度梯度聚丙烯酰胺凝胶电泳（CG-PAGE）和免疫印迹方法鉴定纯化抗原的纯度和特异性。结果亲和层析法纯化抗原经CGPAGE显示5条蛋白带，免疫印迹试验鉴定均有抗原性，强阳性带蛋白分子质量单位为31ku，各区带蛋白与并殖吸虫、血吸虫血清抗体无交叉反应。粗抗原有16条蛋白带。结论经亲和层析法纯化的华支睾吸虫抗原特异性高。亲合层析是获取华支睾吸虫诊断用抗原的较佳方法。     

A族链球菌表面蛋白Fba的原核表达及免疫原性分析

目的 研究A族链球菌(GAS)表面新型纤连蛋白Fba的免疫原性,探讨GAS感染机体的免疫应答机制.方法 PCR扩增fba基因,测序正确后克隆至原核表达质粒pGEX4T-2,并在E.coli BL21中表达,应用ELISA和Western印迹法鉴定目的 蛋白表达,以该蛋白作为抗原,包被酶联板,检测GAS全菌免疫鼠血清、33份抗链球菌"O"阳性患者血清.同时,将该蛋白免疫Balb/C小鼠,3次免疫后收集皿清,检测IgG效价.结果 ELISA和Western印迹证实,表达的Fba重组蛋白可与已知兔抗Fba阳性血清产生特异性反应;且Fba蛋白可与GAS全菌免疫鼠血清、抗链球菌"O"阳性患者血清发生特异性结合.Fba蛋白免疫小鼠后抗血清IgG效价达1:4800.结论 GAS感染或Fba蛋白免疫动物后均可诱导动物体产生Fba抗体,该抗体与Fba重组蛋白可产生特异性反应,提示Fba蛋白具有良好的免疫原性和抗原性.Fba蛋白可作为GAS的候选疫苗及检测GAS感染患者血清效价的重要工具.     

胶体金免疫层析法快速检测鼠疫耶尔森菌F1抗体

目的建立检测鼠疫耶尔森菌F1抗体的胶体金标记免疫层析方法。方法胶体金标记纯化鼠疫F1抗原,双抗原夹心法原理制成免疫层析检测试纸条,并对兔、羊、人和黄鼠血清进行检测评价。结果用80份不同动物种属的阴性血清进行检测,未出现非特异性反应,对5份恢复期病人血清、13份免疫兔血清和2份免疫羊血清检测也取得阳性结果,对于8份I HA临界阳性(滴度1∶20)的达乌尔黄鼠血清检出阳性6份。结论建立了可以用于现场快速检测鼠疫F1抗体的胶体金免疫层析方法。     

丙型肝炎病毒F蛋白抗原性及患者血清F抗体流行率的研究

目的探讨丙型肝炎病毒(HCV)F蛋白的抗原性及患者血清F抗体的流行率。方法利用11条引物延伸获处HCV f基因,以此为模板经聚合酶链反应扩增得到截短型f65基因片段,定向克隆至pET32a(+),重组子转化大肠杆菌Plyss菌株,异丙基β半乳糖苷诱导后用镍离子树脂纯化HCV F65蛋白。以F65蛋白为抗原,酶联免疫吸附法检测HCV感染者血清中F抗体。应用F65蛋白免疫新西兰大白兔制备多克降F抗体,再用葡萄球菌A蛋白树脂纯化兔血清中F抗体。结果成功构建了pET32a(+)f65重组子,表达并纯化了分子量为3．2×10~4的HCV F65蛋白。30份HCV患者血清F抗体的A_(450)为0．125+0．061．F抗体阳性率为63．3%。获得了兔源性F抗体,该抗体与HCV F65蛋白发生特异性结合反应,滴度为1：30000。结论HCV F65蛋白具有抗原性,可用来检测血清中F抗体。HCV患者血清中存在F抗体。兔源性F抗体可用来检测HCV F蛋白。     

应用链球菌菌壁多糖抗体诊断不典型风湿热

目的近年研究发现不典型风湿热病例日渐增多,本研究旨在探讨A组链球菌菌壁多糖抗体(antibodytogroupAstreptococcalpolysaccharide,ASP)对诊断不典型风湿热的价值。方法不典型风湿热组57例,对照组为其他病种患者57例。以亚硝酸法提取A组链球菌菌壁粗多糖,分子筛凝胶层析法分离和纯化,经抗原性检测得到具有抗原活性的相关抗原,并通过酶联免疫吸附试验(ELISA)法检测血清中抗多糖抗体。同时检测血沉(ESR)、抗DNA酶-B(anti-deoxyribonucleaseB)、抗链球菌溶血素O(ASO)。比较各种检测方法的诊断价值。结果ASP诊断风湿热的敏感性73.7%,特异性76.6%;ESR敏感性47.4%,特异性46.4%;ASO敏感性15.6%,特异性94.6%;抗DNA酶-B敏感性65.4%,特异性44.6%。进一步作ROC曲线分析及判别分析,结果ASP曲线下面积最大,判别系数亦最大。结论ASP对不典型风湿热及风湿活动有较好的诊断价值,其敏感性和特异性优于ESR,在判断链球菌感染方面亦优于ASO和抗DNA酶-B等传统的检测指标。     

多孔性阴离子交换树脂血浆吸附治疗重型肝炎疗效及安全性研究

目的 通过观察多孔性阴离子交换树脂血浆吸附在慢重肝救治中的应用,探讨其在重肝退黄及改善症状等方面的应用价值.方法 治疗组30例慢重肝(乙型)病人均经3次多孔性阴离子交换树脂血浆吸附治疗,比较治疗前后症状改善、黄疸消退及反弹、血常规、凝血指标的变化,不良反应的发生等.对照组30例同类病人,仅给予一般内科综合治疗.结果 多孔性阴离子交换树脂血浆吸附治疗.对改善症状部分有效,且无因治疗而引发新的不适.每次治疗对胆红素的吸附效能高.虽然治疗后反弹幅度较大.但总体退黄作用较好.对血白蛋白、血小板、血红蛋白等有一定的影响,但总体不良反应少,具有较好的安全性.结论 多孔性阴离子交换树脂血浆吸附治疗在慢重肝(乙型)病人的救治中,具有较好的应用价值.是重肝治疗方案中的新选择.     

Role of lipoteichoic acid in infection and inflammation

Lipoteichoic acid (LTA) is a surface-associated adhesion amphiphile from Gram-positive bacteria and regulator of autolytic wall enzymes (muramidases). It is released from the bacterial cells mainly after bacteriolysis induced by lysozyme, cationic peptides from leucocytes, or beta-lactam antibiotics. It binds to target cells either non-specifically, to membrane phospholipids, or specifically, to CD14 and to Toll-like receptors. LTA bound to targets can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon. It also triggers the release from neutrophils and macrophages of reactive oxygen and nitrogen species, acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, and cytotoxic cytokines, which may act in synergy to amplify cell damage. Thus, LTA shares with endotoxin (lipopolysaccharide) many of its pathogenetic properties. In animal studies, LTA has induced arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, and periodontal lesions, and also triggered cascades resulting in septic shock and multiorgan failure. Binding of LTA to targets can be inhibited by antibodies, phospholipids, and specific antibodies to CD14 and Toll, and in vitro its release can be inhibited by non-bacteriolytic antibiotics and by polysulphates such as heparin, which probably interfere with the activation of autolysis. From all this evidence, LTA can be considered a virulence factor that has an important role in infections and in postinfectious sequelae caused by Gram-positive bacteria. The future development of effective antibacteriolitic drugs and multidrug strategies to attenuate LTA-induced secretion of proinflammatory agonists is of great importance to combat septic shock and multiorgan failure caused by Gram-positive bacteria.     

Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus

Lipoteichoic acid (LTA), a glycerol phosphate surface polymer, is a component of the envelope of Gram-positive bacteria. However, the molecular basis for its synthesis or function is not known. Here we report that Staphylococcus aureus LtaS synthesizes glycerol phosphate LTA. Construction of a mutant S. aureus strain with inducible ltaS expression revealed that LTA synthesis is required for bacterial growth and cell division. An ltaS homologue of Bacillus subtilis restored LTA synthesis and the growth of ltaS mutant staphylococci. Thus, LtaS inhibition can be used as a target to treat human infections caused by antibiotic-resistant S. aureus or other bacterial pathogens.     

Structure-based mechanism of lipoteichoic acid synthesis by Staphylococcus aureus LtaS

Staphylococcus aureus synthesizes polyglycerol-phosphate lipoteichoic acid (LTA) from phosphatidylglycerol. LtaS, a predicted membrane protein with 5 N-terminal transmembrane helices followed by a large extracellular part (eLtaS), is required for staphylococcal growth and LTA synthesis. Here, we report the first crystal structure of the eLtaS domain at 1.2-Å resolution and show that it assumes a sulfatase-like fold with an α/β core and a C-terminal part composed of 4 anti-parallel β-strands and a long α-helix. Overlaying eLtaS with sulfatase structures identified active site residues, which were confirmed by alanine substitution mutagenesis and in vivo enzyme function assays. The cocrystal structure with glycerol-phosphate and the coordination of a Mn²⁺ cation allowed us to propose a reaction mechanism, whereby the active site threonine of LtaS functions as nucleophile for phosphatidylglycerol hydrolysis and formation of a covalent threonine–glycerolphosphate intermediate. These results will aid in the development of LtaS-specific inhibitors for S. aureus and many other Gram-positive pathogens.     

双歧杆菌脂磷壁酸延缓细胞衰老的作用机制

目的 探讨双歧杆菌脂磷壁酸(Lipoteichoic acid,LTA)在延缓H2O2诱导细胞衰老中的作用.方法 H2O2诱导WI-38细胞衰老,β-半乳糖苷酶细胞化学染色计算衰老细胞百分率变化,RT-PCR和Western印迹检测衰老细胞p21、细胞周期蛋白E(cyclin E)和周期蛋白依赖性蛋白激酶2(CDK2)表达水平的变化.结果 双歧杆菌LTA处理后,β-半乳糖苷酶细胞化学染色阳性细胞百分率较衰老模型组降低(P＜0.01).与年轻对照组相比,衰老模型组细胞中p21的表达增高,cyclin E和CDK2表达降低,而双歧杆菌LTA能够逆转上述变化(P＜0.01).结论 双歧杆菌能延缓H2O2诱导的细胞衰老,机制可能与改变p21,cyclin E和CDK2表达水平有关.     

Bifidobacterium lipoteichoic acid and false ELISA reactivity in aspergillus antigen detection

A major difficulty with the detection of circulating galactomannan, a cell-wall polysaccharide released by Aspergillus sp during growth, in the serodiagnosis of invasive aspergillosis is the occurrence of false-positive ELISA results, especially in neonates and infants. On the basis of molecule similarity, we postulate that a lipoteichoic acid of Bifidobacterium sp can act as epitope for the monoclonal antibody used in the ELISA. The neonatal gut is heavily colonised with Bifidobacterium sp and these bacteria or their lipoteichoic acid might cause ELISA reactivity with serum after translocation because of immaturity of the intestinal mucosa. If our hypothesis is correct, we might find a method to discriminate between false-positive and true-positive ELISA results and thereby prevent unnecessary pre-emptive treatment of patients.     

Abating colon cancer polyposis by Lactobacillus acidophilus deficient in lipoteichoic acid

An imbalance of commensal bacteria and their gene products underlies mucosal and, in particular, gastrointestinal inflammation and a predisposition to cancer. Lactobacillus species have received considerable attention as examples of beneficial microbiota. We have reported previously that deletion of the phosphoglycerol transferase gene that is responsible for lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus (NCK2025) rendered this bacterium able to significantly protect mice against induced colitis when delivered orally. Here we report that oral treatment with LTA-deficient NCK2025 normalizes innate and adaptive pathogenic immune responses and causes regression of established colonic polyps. This study reveals the proinflammatory role of LTA and the ability of LTA-deficient L. acidophilus to regulate inflammation and protect against colonic polyposis in a unique mouse model.     

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid

Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile-induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4(+) T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4(+)CD45RB(high)T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4(+)FoxP3(+) T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.     

Interaction of group A streptococcal lipoteichoic acid with bovine myelin basic protein.

Group A streptococcal lipoteichoic acid (LTA), a polyglycerol phosphate ester linked with fatty acids, formed precipitin lines in agar gels with the positively charged myelin basic protein (MBP) isolated from bovine brain. The LTA-MBP complexes were formed with deacylated teichoic acid and were not inhibited by monomeric glycerol phosphate, a finding indicating the participation of the polyglycerol phosphate (PGP) backbone of LTA in the reaction. Following interaction with PGP, MBP inhibited the agglutination of LTA-sensitized erythrocytes by induction of antibodies to LTA, directed against the PGP antigenic determinant. The amplification of the humoral immune response to the insoluble complex of LTA and MBP was of particular interest in view of the lack of immunogenicity of either LTA or MBP alone.