肺炎嗜衣原体Cpn0425重组蛋白的克隆与表达及抗原性研究

目的构建重组质粒pGEX6p-2/Cpn0425,在E.coli BL21中进行诱导表达,纯化获得重组融合蛋白Cpn0425,并评价其抗原性。方法 PCR扩增肺炎嗜衣原体Cpn0425蛋白编码基因,构建pGEX6p-2/Cpn0425重组质粒,测序正确后在E.coli BL21中诱导表达,用SDS-PAGE和Western-Blot进行分析和鉴定。结果构建了重组质粒pGEX6p-2/Cpn0425,并在E.coli BL21菌中高效表达出Mr约为50kDa的GST-Cpn0425目的蛋白,超声裂解后SDS-PAGE分析目的蛋白在菌体细胞内以可溶性形式表达,经采用默克Novagen的GST纯化树脂纯化获得纯度在95%以上的重组蛋白。结论获得了Cpn0425基因片段,并在E.coli BL21中成功表达,GST-Cpn0425具有良好的抗原性。     

应用直接免疫荧光法快速检测肺炎衣原体

肺炎衣原体是新近命名的新型衣原体,近几年的研究表明,肺炎衣原体是引起人呼吸道感染,特别是肺炎的致病微生物之一。由于肺炎衣原体分离培养困难,目前常用于临床的血清微量免疫荧光法还存在技术上的问题很难标准化,因此关于肺炎衣原体的流行病学、临床及实验室检测方法等,国内尚少有关的研究报道。我们应用单克隆抗体直接免疫荧光法(DFA)检测临床标本112例,旨在调查呼吸道感染者中肺炎衣原体感染情况,现将检测结果报告如下。材料和方法　　一、临床资料　　1994年10月～1995年3月,采集我院呼吸系疾病门诊患者54例,住院患者58例,共计112例…     

肺炎衣原体包涵体核酸组分衍化分析法的建立

吖啶橙（ＡＯ）染色可以作为肺炎衣原体（Ｃｐｎ）包涵体的染色方法。它是一种快速检查Ｃｐｎ感染的较好方法，有可能替代荧光单抗染色的功能，克服单抗染色时细胞结构显示不清的缺点，具有简捷、价廉和既有反映衣原体包涵体的形态和成份，又能反映包涵体不同时期ＲＮＡ和ＤＮＡ含量变化的独特优点。     

肺炎衣原体感染与冠心病关系的临床研究

应用酶联免疫吸附试验检测 12 0例冠心病 (ＣＨＤ)患者血清肺炎衣原体 (Ｃｐｎ)特异性抗体ＩｇＭ、ＩｇＧ及ＩｇＡ ,结合同期血脂水平 ,以探讨Ｃｐｎ感染与ＣＨＤ的关系。一、资料与方法⒈研究对象 :ＣＨＤ患者 12 0例 ,男 6 9例 ,女 5 1例 ,年龄34～ 80 (6 1.86± 12 .5 6 )岁。急性心肌梗死 (ＡＭＩ) 2 6例 ,不稳定性心绞痛 (ＵＡ) 4 2例 ,慢性冠心病 (ＣＣＨＤ) 5 2例 ,ＵＡ及ＣＣＨＤ除部分陈旧性心肌梗死患者外均经冠状动脉造影证实至少 1支冠状动脉狭窄≥ 5 0 %。对照组 111例 ,男 5 4例 ,女 5 7例 ,年龄 41～ 80 (5 6 .5 8± 10 .10 …     

肺炎嗜衣原体Cpn0797蛋白的生物信息学分析及其单克隆抗体的制备和鉴定

摘 要： 目的 生物信息学分析肺炎嗜衣原体Cpn0797蛋白的结构,制备特异性的抗Cpn0797蛋白的单克隆抗体。 方法 用ProtParam、SignalP、NPS@ 、和PSORT等软件对Cpn0797蛋白的理化参数、信号肽、二级结构、蛋白亚细胞定位进行分析;原核表达纯化GST-Cpn0797融合蛋白,以其为免疫原免疫BALB/c小鼠,杂交瘤技术制备单克隆抗体,采用间接免疫荧光法鉴定单克隆抗体的亚类和特异性。 结果 生物信息学分析表明Cpn0797蛋白二级结构以无规则卷曲为主;成功地建立了能稳定分泌抗Cpn0797蛋白的单克隆抗体杂交瘤细胞株,单克隆抗体能特异性的识别Cpn0797内源性蛋白。结论 成功制备特异性的Cpn0797单克隆抗体,为进一步探究Cpn0797蛋白的生物学功能提供实验基础。     

600例呼吸道感染肺炎支原体和嗜衣原体感染状况的分析

目的 探讨儿童呼吸道感染者肺炎支原体(Mp)、肺炎嗜衣原体(Cp)的感染情况.方法 Mp检测采用聚合酶链反应(PCR)和培养法,Cp检测采用微量免疫荧光试验对600例呼吸道感染患儿进行检测,并以健康体检的300例健康儿童作为对照.从感染率、感染年龄等给予分析.结果 观察组总感染率、Mp、Cp单项感染率均高于对照组(P＜0.01);＞3岁组Mp、(Mp+ Cp)及总感染率均显著高于≤3岁组(P＜0.01),两组的Cp感染率亦有显著差异(P＜(.05).结论 儿童呼吸道感染患者有较高的Mp及Cp感染率,Mp感染和(Mp+Cp)感染以＞3岁儿童为主,应引起儿科医师的高度重视.     

肺炎嗜衣原体感染与动脉粥样硬化的关系

本文介绍了肺炎嗜衣原体感染与动脉粥样硬化的关系、肺炎嗜衣原体在体内的传播及其与血管内皮细胞、单核/巨噬细胞、平滑肌细胞之间的相互作用,以及肺炎嗜衣原体感染对动脉粥样硬化斑块稳定性的影响和肺炎嗜衣原体感染致动脉粥样硬化的机制。     

核酸扩增技术在肺炎嗜衣原体感染诊断中的应用

肺炎嗜衣原体(Chlamydophila pneumonia,Cpn)是一类引起人类呼吸道感染的、专营真核细胞内寄生生活的微生物,在人体中存在持续感染.Cpn感染早期的快速诊断有利于阻止疾病的传播和防止并发症的形成.有关Cpn的诊断方法,国内外学者进行了一系列研究并取得了很大的进展.本文就核酸扩增技术在诊断Cpn感染中的应用作一综述.     

脑卒中与肺炎衣原体感染的相关性

目的探索肺炎衣原体(Cpn)慢性感染与脑卒中之间的关系。方法2002年3月-2006年1月来复旦大学附属金山医院就诊的25岁至72岁脑卒中患者60例为病例组,选择60名性别、年龄和种族等与病例组相匹配的无脑卒中者为对照组,用微量免疫荧光法检测血清中Cpn抗体(IgA、IgG),并比较血清总胆固醇(TC),甘油三酯(TG)水平的差异。结果在脑卒中组和对照组血清中,Cpn IgA的阳性检出率分别为75.0%(45/60)和38.3%(23/60),P<0.001;Cpn IgG的阳性检出率分别为58.3%(35/60)和43.3%(26/60),P>0.05。脑卒中组TC(5.75±0.74 mmol/L)高于对照组(4.62±0.76mmol/L),具有统计学意义(P<0.05),TG水平差别无统计学意义。结论肺炎衣原体慢性感染的血清学证据与脑卒中危险有关,IgA滴度比IgG可能是更好的危险标记物。这一发现表明,脑卒中与Cpn之间有着临床的紧密关联,有关病因学的关系,有待进一步研究。     

呼吸道感染患者外周血中肺炎嗜衣原体的检测与分析

目的检测并分析呼吸道疾病患者的肺炎嗜衣原体（Cpn）感染率。方法收集173例呼吸道感染患者和52例健康体检者外周血标本,分离外周血单核细胞（PBMC）血清。提取纯化PBMC的DNA,以该DNA为模板,用特异性引物体外扩增CpnMOMP基因。扩增产物经电泳及测序鉴定,确认为Cpn特异性的目的基因。用ELISA法检测待测血清中的Cpn-IgG抗体。通过PCR与ELISA的检测结果,结合临床资料进行流行病学综合分析Cpn感染率。结果以PBMC基因组DNA为模板,呼吸道感染组患者标本中CpnMOMP阳性率为32．40％（56／173）,健康对照组中阳性率为9．62％（5／52）;经测序比对,所有MOMP阳性扩增产物与Cpn AR-39的MOMP基因同源性在99％以上。ELISA检测血清Cpn-IgG,感染组阳性率50．87％（88／173）,对照组阳性率为30．77％（16／52）,两组差异有统计学意义。比较不同年龄组ELISA及MOMPPCR两种方法检测Cpn感染指标的阳性率,发现老年人的Cpn感染率最高。结论在呼吸道感染患者不同年龄阶段均检出较高的Cpn感染指标,其中老年人群中检出率最高,说明Cpn是本地区呼吸道感染常见的病原体之一。     

肺炎衣原体、支原体在哮喘致病机制中的作用

肺炎衣原体、支原体在哮喘致病机制中的作用引起了国际学者极大的兴趣。随着对肺炎衣原体、支原体与哮喘相关性认识程度的加深,结合众多学者所做的富有成效的研究成果,现将肺炎衣原体、支原体感染在哮喘致病机制中作用的研究进展作一综述。     

肺炎支原体重组蛋白抗原与膜蛋白抗原诊断试剂在临床应用中的比较

目的以肺炎支原体（Mp）重组蛋白做包被抗原检测临床呼吸道感染患儿血清标本,验证重组蛋白抗原的诊断价值。方法利用Mp重组蛋白抗原包被酶标板,ELISA法检测临床疑似Mp感染患儿的血清标本,同时用Mp膜抗原被动凝集试验平行检测,并对比分析两种方法的检测结果。结果重组蛋白抗原ELISA检测Mp抗体的阳性率为49.00%（98/200）,膜抗原被动凝集试验Mp抗体的阳性率为63.50%（127/200）。两种检测方法的总符合率为70.50%。阳性符合率为58.45%。结论Mp重组蛋白抗原ELISA检测肺炎支原体感染有诊断价值,其特异性优于膜抗原被动凝集试验。     

慢性肺炎衣原体感染是慢性阻塞性肺病（COPD）的危险因子

目的研究慢性Cpn感染和慢性阻塞性肺病（COPD）之间可能的相关性。方法观察组为年龄在58岁以上的轻度至重度的COPD患者165例和年龄、性别匹配的80例对照组,并测定其FEVl、FVC和圣乔治呼吸问卷（SGRQ）计分。用直接免疫荧光（DIF）法检测外周血单核细胞（PBMC）中的肺炎衣原体特异性抗原（Cpn—Ag）,同时用间接微量免疫荧光（MIF）法检测Cpn抗体（IgA,IgG和IgM）。结果观察组Cpn的感染率为63．0％（104／165）,对照组15．0％（12／80）。代表急性感染的Cpn—Ag和Cpn-IgM抗体的检出率观察组亦显著高于对照组（P〈O．001）,代表慢性感染的Cpn—IgA和IgG抗体的检出率观察组也显著高于对照组（P％0．001）。阿奇霉素治疗后观察组病人的临床症状均有显著改善：SGRQ记分和FEV。／FVC（％）显著增加,与此同时观察组只有Cpn—IgM滴度显著下降（P〈O．001）。慢性Cpn感染与吸烟及较高的年龄有关但与性别无关。结论慢性Cpn感染可能是COPD发展的独立危险因素。     

Acute exacerbation of idiopathic pulmonary fibrosis: role of Chlamydophila pneumoniae infection

BACKGROUND AND OBJECTIVES: Patients with idiopathic pulmonary fibrosis (IPF) may experience acute exacerbations of their illness. The actual trigger(s) of such exacerbations is unknown. Chlamydophila pneumoniae infection can cause exacerbation of asthma and COPD. A prospective study was conducted to investigate the possible role of C. pneumoniae infection in triggering acute exacerbations of IPF. METHODS: A prospective observational study over 5 years of consecutive IPF patients who fulfilled the criteria for acute exacerbation. Sputum, blood cultures and acute and convalescent serology for C. pneumoniae IgG and IgA (ELISA) were performed. RESULTS: Previous infection with C. pneumoniae is common. Of the 27 study patients, 15 had a C. pneumoniae IgG index of 1.10-2.99 (positive) and 3 had a C. pneumoniae IgG index of >2.99 (strongly positive) at the time of presentation with an acute exacerbation. In addition, 15 subjects had a C. pneumoniae IgA index of 1.10-2.99 (positive) and 6 subjects had a C. pneumoniae IgA index of >2.99 (strongly positive). However, only two of the 15 subjects (13%) for whom paired sera were tested exhibited a significant rise in antibody response (change in index of 1.90 for C. pneumoniae IgG and 1.54 for IgA, respectively) indicating either acute or reactivated infection with C. pneumoniae. There were 15 deaths (56%) despite supportive care that included high-dose corticosteroid therapy and oxygen supplementation. CONCLUSIONS: Mortality is high with acute exacerbation of IPF. Acute infection with C. pneumoniae is uncommon at the time of presentation with acute exacerbation of IPF.     

Detection of Chlamydophila pneumoniae in the bone marrow of two patients with unexplained chronic anaemia

Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.     

Human immunodeficiency virus,atherosclerosis and Chlamydophila pneumoniae

Chlamydophila pneumoniae (C. pneumoniae) is an obligate, intracellular bacterium associated with a wide variety of acute and chronic diseases. C. pneumoniae infection is characterized by persistence and immunopathological damage to host target tissues, including the lung. Over the past 20 years, a variety of studies have investigated a possible link between C. pneumoniae infection and atherosclerosis, because of its role in all stages of atherosclerosis, from initial inflammatory lesions to plaque rupture. In the current highly active antiretroviral therapy (HAART) era, many human immunodeficiency virus (HIV)-infected patients are experiencing health problems that accompany the aging process, mainly the risk of cardiovascular disease (CVD). There is renewed interest in a link between atherosclerotic CVD and as yet poorly defined environmental exposures, including infectious agents. On the one hand, the patient with HIV and lipodystrophy caused by HAART and exacerbated by C. pneumoniae infection could be a factor of risk for atherosclerosis. An assessment of the therapy against C. pneumoniae and HAART should always be conducted. It is advisable that HIV-acquired immune deficiency syndrome patients undergo a serological test to determine exposure to C. pneumoniae and to assess treatment options. On the other hand, in patients with a positive serology to C. pneumoniae, an increment of the body mass index has been found; therefore, it is probable that the recurrent infection may play an important role in creating adverse endothelial conditions allowing the infection by C. pneumoniae in its chronic form, to damage the endothelial surface. Vascular studies would be necessary for corroboration.     

Evaluation of enzyme-linked immunosorbent assay for Chlamydophila pneumoniae-specific immunoglobulin M in acute respiratory tract infection

Background and objective:   The study evaluated a newly developed ELISA (Hitazyme Chlamydophila pneumoniae ) for detecting anti- C. pneumoniae -specific IgM antibody, by comparing the ELISA assay to a microimmunofluorescence (MIF) test and immunoblotting.
Methods:   One hundred patients with acute respiratory tract infections (58 children and 42 adults) were enrolled in the study. Paired sera were obtained from all subjects for serological testing of C. pneumoniae .
Results:   C. pneumoniae IgM positivity was observed in 36 (62.0%) children and 11 (26.1%) adults. However, MIF test or immunoblot revealed only four positive reactions in these patients. These four IgM-positive patients were also positive by ELISA. A significant increase in IgG and/or IgA antibody titres in paired sera was observed in three of the four patients. Of the remaining 96 patients, no significant increase in IgG or IgA antibody titre in the paired sera was observed. To confirm the positive reactivity of ELISA, positive sera were also analysed by recombinant enzyme immunoassay. Forty-three cases that were IgM-positive only by ELISA were all negative by recombinant enzyme immunoassay and the ELISA results were considered to be false-positives.
Conclusions:   These results indicate that a newly developed ELISA for detecting anti- C. pneumoniae -specific IgM antibody frequently generates false-positive findings in patients with acute respiratory tract infections, at the current cut-off level. Further studies are needed to determine an appropriate cut-off level and the possible causes of the false-positive results in the ELISA.     

Comparison of serological tests for detection of immunoglobulin M antibodies to Chlamydophila pneumoniae

Background and objective:   To evaluate an enzyme immunoassay (EIA) (AniLab C. pneumoniae ) for detecting anti- Chlamydophila pneumoniae -specific IgM antibody, by comparing it with an ELISA, Hitazyme C. pneumoniae , and a micro-immunofluorescence (MIF) test.
Methods:   Antibodies in sera from three groups of patients were measured: eight serum samples collected serially from a patient with acute C. pneumoniae pneumonia, 34 serum samples with Hitazyme-ELISA false-positive results, and 137 serum samples from patients with community-acquired pneumonia.
Results:   The IgM antibody titre in the patient with acute C. pneumoniae pneumonia showed almost identical variation with the EIA, ELISA and MIF tests. Among the 34 samples found to be false-positive for IgM with ELISA, EIA revealed no positive cases. When a true positive case was defined as one for which a positive reaction was obtained with at least two tests, the sensitivities of the EIA, ELISA and MIF tests were 97.1%, 100% and 74.3%, with specificities of 100%, 37.3% and 100%, respectively.
Conclusions:   EIA was highly sensitive and specific as compared with the MIF test, and the ELISA test showed the lowest specificity. Consequently, the AniLab-EIA, rather than the Hitazyme-ELISA, is recommended as the routine method for accurately diagnosing acute C. pneumoniae infection.     

基于A1E3及BIC4单克隆抗体检测日本血吸虫循环抗原ELISA法的建立及现场初步应用

目的建立基于AlE3及B1C4单克隆抗体检测日本血吸虫循环抗原的夹心酶联免疫吸附试验（ELISA）,并对其应用情况进行初步评价。方法采用十二烷基磺酸钠一聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹试验（Westernblot-ring）对A1E3及B1C4单克隆抗体进行特性分析,用ELISA法测定B1C4、A1E3单克隆抗体效价。采用棋盘格滴定法确定双单抗夹心ELISA法检测循环抗原的最佳工作浓度。在最佳条件下,分别检测20份急性血吸虫病病人血清、46份慢性m吸虫病病人血清及20份止常人血清,评价其检测敏感性和特异性。用建立的双单抗夹心ELISA法和市售检测血吸虫循环抗原的ELISA试剂盒检测湖北省江陵县IHA阳性血吸虫病病人血清72份,以评估双单抗夹心ELlSA法的检测效能。结果经SDS-PAGE和Westernblotting分析,纯化后的A1E3及B1C4在相对分子质量（M,）88000和52000处各有一条清晰重链,在吖．20000处有一条相同的轻链,且A1E3及B1c4可与可溶性虫卯抗原（SEA）及急性血口发虫病病人血清发牛特异性反应。BlC4单抗效价可达1：10^5,A1E3单抗效价可达1：30000。用B1C4、A1E3双单抗夹心ELISA法检测急、慢性血吸虫病病人粗清阳性率分别为100％和86．9％,检测20份正常人血清特异性为100％。双单抗夹心ELISA法及市售EIJJsA试剂盒检测日本血吸虫循环抗原阳性率分别为45．8％及43．1％。结论成功建立了基于A1E3及B1C4双单抗夹心ELISA法,该法检测日本m吸虫循环抗原具有较高的敏感性及特异性。