肺炎嗜衣原体抗原与血清学检测的研究

【作者】 陈学勤；

【导师】 钱利生； 李雪萍；

【作者基本信息】 复旦大学 ， 病原生物学， 2008， 硕士

【摘要】 肺炎嗜衣原体（Chlamydophila,C.pneumoniae,缩写Cpn）是一种常见的呼吸道病原体,所致疾病最常见的是肺炎和支气管炎,近年来诸多研究表明其与动脉粥样硬化综合症,哮喘等疾病有关。由于Cpn为严格胞内寄生性,分离培养较为困难,从而影响深入研究Cpn的致病机制、特异性诊断和疫苗的开发。本文应用进口Cpn菌株,通过建立Cpn感染细胞技术,获取大量Cpn抗原,制备斑点抗原片,用于微量免疫荧光染色（Micro-immunofluorescence,MIF）检测血清抗体。同时根据Cpn主要外膜蛋白（Major outer membrane protein,MOMP）核苷酸序列,构建MOMP/pQE31重组质粒,制备MOMP重组抗原,用于ELISA检测诊断Cpn感染。通过对以上检测结果的分析,较为客观的评定每种方法的血清抗体特异性和敏感性,以便用于辅助Cpn感染的临床诊断,探寻Cpn感染与疾病的因果关系。论文第一部分应用进口Cpn菌株,感染Hep-2细胞,分别以瑞氏-姬母萨染色、吖啶橙（Acridine Orange,AO）染色和直接免疫荧光（Direct Immunofluorescence,DIF）染色三种方法鉴定Cpn感染细胞。Cpn感染Hep-2细胞的最适条件是用含1ug/ml放线菌酮的维持液,在35℃5%CO₂孵箱中培养7天,并在培养的第0,3,4,5天作2600rpm/min离心1h,感染成功率极高。染色反应显示,瑞氏-姬母萨染色可将Cpn包涵体染成蓝紫色或红紫色;吖啶橙染色则显示Cpn感染的Hep-2细胞呈现鲜明的橘红色;免疫荧光抗体染色后,在Cpn感染细胞内可见亮苹果绿色包涵体。论文第二部分是将Cpn抗原用于制备斑点抗原片。通过建立MIF检测血清抗体,诊断Cpn感染。用Cpn抗原片和外周血单核细胞（Peripheral BloodMononuclear Cell,PBMC）抗原片分别检测100份病人血液标本。结果显示:用Cpn抗原片检测血清IgG抗体,阳性率为61%;用PBMC抗原片检测,阳性率60%。经统计,两者阳性检出率无明显差异。用Cpn感染细胞制作的Cpn斑点抗原片可用于临床检测血清Cpn-IgG抗体,且具有特异性、敏感性高的特点。论文第三部分通过构建和表达Cpn MOMP重组抗原,应用包涵体纯化法纯化重组抗原后建立ELISA试验,并检测了82份正常人血清及临床标本207份,其中心血管疾病血清118份,呼吸系统疾病血清59份,其他疾病30份。用重组抗原检测血清IgG,正常人血清阳性检出率仅为3.7%;而207份病人血清,116份为阳性,总阳性率56.0%。其中心血管疾病血清118份,阳性69份,阳性率58.5%;呼吸系统疾病血清59份,阳性38份,阳性率64.4%;其他疾病患者30份,阳性9份,阳性率仅30.0%。与进口ELISA试剂盒及Cpn全抗原相比,阳性率基本相符,统计分析无显著意义,但重组MOMP蛋白的抗原特异性较高,且具有抗原制备较为方便和价廉等特点。本课题通过细胞培养技术制备获得大量Cpn,经瑞氏-姬母萨染色、吖啶橙染色、免疫荧光抗体染色鉴定证实为Cpn抗原。将纯化的Cpn抗原制成抗原片,通过Cpn抗原片与PBMC抗原片检测血清抗体的比较,经统计显示阳性检出率无明显差异。表明Cpn抗原片检测法具有较好的敏感性和特异性,可用于临床辅助诊断。制备重组MOMP抗原用于ELISA检测血清抗体,结果显示正常人及病人血清抗体效价有明显差异,且与进口试剂盒及全抗原检测阳性率基本相符。以重组蛋白MOMP为基础建立的ELISA方法灵敏度和特异性高,可用于Cpn感染的诊断和所致疾病的研究,具有重要的临床意义和学术价值。更多还原

【Abstract】 Chlamydophila pneumoniae (Cpn), which belongs to the chlamydiae, is an important respiratory tract pathogenic microorganism. The most common deseases caused by Cpn are pneumoniae and bronchitis, recent researches also indicated its relationship with artherosclerosis desease, bronchial asthma, sarcoidosis and asthma. Cpn is proved to be difficult to isolate and culture, which limited carrying out further study about it’s pathogenic mechanism, specific diagnosis and vaccine exploitation. Imported Cpn strain was used to set up the model of Cpn infected cell, so as to obtain a great quantity of Cpn antigen to prepare spot antigen slide. These slides were used to estabilish Micro-immunofluorescence (MIF) method to detect serum antibody. According to the sequence of Major outer membrane protein (MOMP) , we also established a recombined plasmid of MOMP/pQE31 and producted recombined Cpn MOMP antigen to detect serum IgG. Through the comparison of the results of these methods, we expected to evaluate each method’s specificity and sensitivity so that to help the clinical diagnosis to the Cpn infection and analyze the relationship between its infection and diseases.In the first part of this paper, imported Cpn strain was used to infect Hep-2 cell and the result of infection was identified trough Romanowsky-Giemsa Staining, Acridine Orange(AO) Staining and Direct Immunofluorescence (DIF) Staining. Large amount of Cpn antigen was obtained after purification. It showed that the optimization culture of Cpn was in Hep-2 with 1640 added 1ug/ml cycloheximide and centrifugated on days 0, 3, 4, and 5, and kept culturing for 7 days. Thus can make a really high infection ratio. It also showed Cpn infected Hep-2 cell would turn amethyst or magenta using Romanowsky-Giemsa Staining, bright orange using AO Staining and apple green cytorrhyctes using DI staining.In the second part of this paper, Cpn antigen was used to prepare Cpn spot antigen slide. We established the method of detecting serum Cpn antibody and diagnosing Cpn infection through MIF. One hundred sera of patient tested with Cpn spot antigen slide as well as Peripheral Blood Mononuclear Cell (PBMC) spot antigen slide. Cpn antigen slide got the Cpn -IgG positive rate of 61 % while the PBMC antigen slide was found to have a positive rate of 60%. Statistical analysis show that there is little difference between the positive rate of the two methods. Cpn spot antigen slide which made by Cpn infected cells is proved to be both highly specific and quite sensitive and can be use to detect serum Cpn-IgG antibody.In the third part of this paper, by establishing and expressing recombined Cpn MOMP antigen, which was purified and used as antigen in ELISA assays. Sera were obtained from 82 healthy volunteers with 207 patients. The patient samples consisted of 118 sera with cardiovascular diseases, 59 sera with diseases of respiratory system and 30 sera with other diseases. Cpn IgG-positive results were observed in 56.0% of patients and only in 3.7% of healthy subjects. There were 69 positive sera of 118 with cardiovascular diseases, 38 positive of 59 with diseases of respiratory system and 9 positive of 30 with other diseases. The positive rates respectively were 58.5%, 64.4% and 30.0%.The positive rate of recombined Cpn MOMP antigen were consistent with the ELISA kit and Cpn holoantigen.In this research, imported Cpn strain was used to infect Hep-2 cell and the result of infection was identified through Romanowsky-Giemsa Staining, AO Staining and DIF Staining. After purification, large amount of Cpn antigen was used to prepare Cpn spot antigen slide. Through comparison between the two method of Cpn spot antigen slide and PBMC antigen slide detecting serum IgG, little difference was shown between the positive rate of the two methods. Cpn spot antigen slide is proved to be both highly specific and quite sensitive and can be use to detect serum Cpn-IgG antibody. Recombined Cpn MOMP antigen was expressed, purified and used as antigen in ELISA assays, which can easily tell the difference between the health and the patient serum. Besides, the positive rate of recombined Cpn MOMP antigen were consistent with the ELISA kit, which showed to be highly specific and sensitive and could greatly help the investigation of Cpn infection. It also had considerable theoretical and pratical value.更多还原