【作者】 贾天军；

【导师】 刘殿武； 钟光明；

【作者基本信息】 河北医科大学 ， 病原生物学， 2008， 博士

【摘要】 肺炎衣原体是一类严格的活细胞内寄生的、具有独特生活周期的微生物。其复制和生物合成是在被感染的宿主细胞内形成的包涵体中进行的,因此包涵体是衣原体赖以生存和繁殖的微环境,为了建立和维持衣原体在感染细胞包涵体内的生长,就必须通过包涵体膜与宿主细胞进行物质和信号的交换。自从1995年Rochey等用实验首次证实衣原体通过自身表达的蛋白而主动修饰包涵体膜以来,陆续有新的包涵体膜蛋白被鉴定出来,并对部分包涵体膜蛋白的生物学作用进行了研究,初步显示包涵体膜蛋白参与与宿主间的相互作用及其致病性。由于包涵体膜蛋白在研究衣原体中发育以及致病过程中的重要地位,寻找并探究包涵体膜蛋白的生物学作用成为研究的热点之一。根据已经鉴定的膜蛋白的疏水性特征,计算机辅助程序预测出90个肺炎衣原体(CPn)基因编码的蛋白为候选包涵体膜蛋白,但目前通过直接抗体标记检测证实的包涵体膜蛋白为数不多。已有的发现提示并非所有计算机预测的包涵体膜蛋白均位于包涵体膜上。因此使用试验方法发现和确认假定的包涵体膜蛋白并进一步探讨其特性对深入了解衣原体的生物学作用、致病性以及预防具有重要意义。CPn0308是预测的包涵体膜蛋白,是否定位于包涵体膜上尚有待于进一步确认。为此本研究在检索CPn基因库信息的基础上,分析CPn0308及其相关基因的特性;克隆CPn0308及其相关基因并表达其GST融合蛋白;制备多克隆和单克隆抗体,对内源性蛋白的表达进行定位;在定位的基础上对其特性进一步研究,以期为更全面了解衣原体包涵体膜蛋白的生物学作用提供信息。第一部分CPn0308及其相关基因的克隆与表达目的:分析和克隆CPn0308和与其相关的其他衣原体基因(包括CT249、MoPn052和GPIC0474)的基础上,进一步表达相关的蛋白,为后续制备抗体、鉴定包涵体膜蛋白奠定基础。方法:①依据文献提供的信息,检索CPn0308信息,根据检索提示,进行相关分析。②选择肺炎衣原体CPn0308基因的开放读码框架区的基因和相关基因CT249、MoPn0520、GPIC0474以及所用的克隆和表达载体pGEX-6P2设计引物;采用PCR方法获取目的基因。③采用常规的酚-氯仿法提纯扩增的目的基因,进行限制性内切酶酶切处理。④用T4连接酶连接纯化后的消化产物与预处理的pGEX-6P2载体,然后转化感受态细胞XL1-blue,接种在含有100μg/ml AMP的选择性培养基LB平板上,37℃过夜培养。⑤采用针对pGEX-6P2载体的PCR初步筛选选择性培养基上生长的克隆;对PCR扩增阳性的克隆提取质粒,进一步采用交叉-PCR进行鉴定;交叉PCR扩增阳性的质粒进行测序并进行序列分析。⑥用IPTG诱导序列正确的克隆表达GST重组蛋白,用Glutathione Sepharose TM 4B纯化重组蛋白,SDS-PAGE凝胶电泳鉴定。结果:①CPn0308基因长度为366bps,编码121个氨基酸残基,分子量为12.971kDa。CT249、GPIC0474和MoPn0520在基因结构与CPn0308具有相似性。一个基因独自为一个基因群,两侧均为已知的基因glgP和dnaA,编码的氨基酸残基数相似。②获得了载体重组质粒pGEX-6P2/CPn0308、pGEX-6P2/CT249、pGEX-6P2/MoPn0520和pGEX-6P2 /GPIC0474。经过用针对pGEX-6P2载体的引物初步筛选后进一步提取重组质粒,经过pGEX-6P2的引物和特异性引物进行交叉PCR扩增,获得的扩增产物与预期结果一致。直接以重组质粒pGEX-6P2/CPn0308、pGEX- 6P2/CT249、pGEX-6P2/MoPn0520和pGEX-6P2/GPIC0474为模板进行测序,得到克隆片段的DNA序列,与基因库中的序列比对的一致性均为100%,进一步分析发现均含有两个跨膜区域。③选取阳性克隆诱导表达重组蛋白,经过纯化后进行SDS-PAGE电泳,显示条带与预期结果一致。结论:①CPn0308及其相关的CT249、MoPn0520以及GPIC 0474基因结构相似。②成功地将CPn0308以及CT249、MoPn0520和GPIC0474克隆到pGEX-6P2载体中,并表达出GST-CPn0308等四个GST融合蛋白。第二部分CPn0308、CT249、MoPn0520和GPIC0474抗体制备及应用目的:本研究在第一部分克隆、表达蛋白基础上进一步制备CPn0308及其相关蛋白的多克隆抗体以及CPn0308的单克隆抗体;采用抗体标记检测法对四种内源性蛋白进行初步定位分析,以期为深入探究这些假定蛋白的生物学作用奠定基础。方法:①用Glutathione Sepharose TM 4B纯化融合蛋白GST-CPn 0308、GST-CT249、GST-MoPn0520和GST-GPIC0474作为免疫原,常规方法免疫Balb/C小鼠,分离血清获得多克隆抗体。对GST-CPn0308蛋白,常规方法制备单克隆抗体。②采用IFA进行筛选阳性克隆和效价滴定。③用IFA方法对所获取的单克隆抗体进行类别的鉴定;应用Western Blotting技术进行检测抗体所识别的CPn0308的部位特异性和识别部位的鉴定。④用所制备的抗体进行不同组合的IFA对四种抗体所对应的内源性蛋白进行初步定位。进一步用吸附试验和抗GST-CPn0308的多克隆抗体直接对四个不同种属的衣原体(AR39、L2、MoPn和GPIC)感染后的HeLa细胞进行IFA分析CPn0308与其他三个蛋白的同源性。结果:①IFA测试血清的效价如下:CPn0308、MoPn0520、CT249和GPIC0474分别为1:2000、1:200、1:2000和1:200。②制备CPn0308的单克隆抗体获得四株阳性杂交瘤细胞株,分别命名为2D7、3A6、3H5、5E10;单克隆抗体均为IgG类抗体,其中2D7为IgG3亚类、3A6为IgG1亚类、3H5和5E10均为IgG2b亚类;四株单克隆抗体识别CPn0308的部位均为CPn0308N,即靠近氨基端一侧。③IFA结果显示假定衣原体蛋白CPn0308、CT249和MoPn0520位于包涵体膜上,即包涵体膜蛋白,而GPIC0474则不位于包涵体膜上。④与对照比较,用GST-CPn0308蛋白吸附GST-CPn0308的抗体未见包涵体膜蛋白的染色,而用GST-CT249、GST-MoPn0520和GST-GPIC0474吸附后仍然可见GST-CPn0308的抗体的特异性包涵体膜蛋白的染色。用GST-CPn0308的多克隆抗体直接对不同种属的衣原体感染的HeLa进行抗体染色,GST-CPn0308的多克隆抗体只识别AR39感染所形成的包涵体膜蛋白,而不识别其他三种衣原体感染所形成的包涵体膜及其包涵体内的衣原体。结论:①成功制备出四株CPn0308的单克隆抗体(2D7、3A6、3H5和5E10),均为IgG类,识别CPn0308的氨基端。②用多克隆抗体对相应的蛋白进行定位,假定蛋白CPn0308、CT249、MoPn0520位于各自衣原体感染HeLa细胞形成的包涵体膜上,而假定蛋白GPIC0474则不在包涵体膜上。③试验证实CPn0308与CT249、MoPn0520以及GPIC0474没有同源性。第三部分假定蛋白CPn0308特性的初步研究目的:使用抗体标记技术对假定蛋白CPn0308进行鉴定,并对其特性进行初步研究。方法:①用含有R12AR39和CPn0308的多克隆抗体或单克隆抗体作一抗进行IFA,在荧光显微镜下观察假定蛋白CPn0308在感染细胞内的初步定位。②选用已经鉴定的CPn蛋白CPAFcp、IncA、MOMP和HSP60分别与CPn0308进行共染色。在激光共聚焦显微镜下观察目的蛋白CPn0308与其他参照蛋白的定位情况。③使用脂质体2000转染试剂分别转染重组体pDsRed-C1/CPn0308和pDsRed-C1/CPn0186到HeLa细胞,在荧光显微镜下观察CPn0308抗体的染色特性;通过吸附试验观察CPn0308抗体和IncA抗体对包涵体膜蛋白染色的特异性;采用Western Blotting技术检测CPn0308抗体结合内源性以及外源性蛋白的特异性。④分别在AR39感染HeLa细胞后的不同时间点进行IFA实验,观察内源性CPn0308蛋白的表达时相。结果:①使用融合蛋白GST-CPn0308的多克隆抗体和单克隆抗体检测目的蛋白显示初步定位于包涵体膜上。②选择CPn0308的单克隆抗体与参照蛋白CPAFcp、IncA、MOMP和HSP60进行共染色,所检测到信号表明CPn0308与CPAFcp、MOMP和HSP60没有交叉现象,与IncA有重叠现象,均位于细胞膜上,重叠部分的颜色为黄色。进一步使用激光共聚焦显微镜对CPn0308在包涵体膜上的定位,发现CPn0308的存在模式明显不同于CPAFcp、MOMP和HSP60的模式,染色结果显示绿、红、蓝三种颜色。但与IncA有重叠现象,颜色显示为蓝、黄两种颜色。即使从Z轴不同的聚焦点观察,均有与IncA重叠现象。③当用针对CPn0308的多克隆抗体和单克隆抗体对转染的细胞进行染色时显示,pDsRed-C1- CPn0308转染HeLa细胞内表达的蛋白染色为黄色,而pDsRed-C1-CPn 0186转染HeLa细胞内表达的蛋白染色为红色;当用针对CPn0186的单克隆抗体进行染色时,正好相反。与对照相比较,当CPn0308的抗体用GST-CPn0308预吸附后染色可见抗体检测信号消失,而用GST-CPn0186预吸附后染色仍然可见;当CPn0186的抗体用GST-CPn0308预吸附后染色可见检测信号,用GST-CPn0186预吸附后信号消失。与参照抗体检测信号相比,Western Blotting显示用CPn0308的抗体只检测到AR39感染后的HeLa细胞的裂解液和GST-CPn0308有相应的蛋白条带,而其他泳道没有可检测到的蛋白条带。以上从三个不同的方面证实抗体结合包涵体膜的特异性。④在AR39感染后的不同时间点,进行IFA观察显示在感染后24h可明显观察到内源性的CPn0308蛋白表达在包涵体的膜上,并且在整个感染期间一直存在包涵体膜上。结论:①用CPn0308的多克隆抗体和单克隆抗体首次试验证实假定蛋白CPn0308位于CPn感染HeLa细胞形成的包涵体膜上。②共定位发现CPn 0308在包涵体上分布模式类似于IncA,而不同于参照蛋白CPAFcp、MOMP和HSP60。③CPn0308的表达时相为在感染后24h表达于包涵体膜上,并在后续的感染期间一直存在包涵体膜上。第四部分感染肺炎衣原体人群中CPn0308免疫原性的检测分析目的:在分析缺血性脑血管疾病(ICVD)患者肺炎衣原体感染的基础上进一步分析CPn0308的免疫原性。方法:①收集相关的临床标本,分离血浆和提取外周血中单个核细胞(PBMC)中的DNA。②用肺炎衣原体的IgG和IgM诊断试剂盒检测血浆中肺炎衣原体的感染情况。③用PCR法检测分析外周血单个核细胞中肺炎衣原体DNA的存在情况。④用Western Blotting法分析内源性蛋白CPn0308的免疫原性。结果:①在所检测的70例ICVD标本中,IgG的阳性率为51.43%,与对照比较有统计学意义;IgM的阳性率2.86%;与对照比较,无统计学意义。②ICVD患者CPn DNA的阳性率为81.08%,显著高于对照组。③当血浆标本按1:500稀释后,仍可检测到CPn0308的抗体。当用结合GST-CPn 0308的凝胶微粒(Beads)预吸附可中断反应,而用GST-CPn0186 Beads预吸附则不能中断反应,说明反应的特异性。结论:①ICVD患者CPn IgG的阳性率明显升高。②ICVD患者PBMC中CPnDNA阳性率显著升高;检出率高于EIA法,是一种敏感的检测CPn感染的检测方法。③初步分析显示CPn0308在感染人群中具有一定的免疫原性。更多还原

【Abstract】 Chlamydia pneumoniae are obligate intracellular parasites of eukaryotic cell with a unique biphasic life cycle. Since chlamydial organisms accomplish all their biosynthesis and replication within the cytoplasmic vacuole (designated as inclusion) of the infected cell. In order to maintain a successful intra- vacuolar growth, Chlamydia has to exchange both materials and signals with host cells via the inclusion membrane. Since Rockey et al. identified the first Inc (IncA) in 1995, more Incs have been found and more information on Inc function has been obtained. The studies have demonstrated that Inc proteins can actively interact with the host cells, potentially benefiting C. pneumoniae growth and contributing to C. pneumoniae pathogenesis.Searching for novel Inc proteins may help unravel the molecular basis of chlamydial interactions with host cells and has thus become a hot topic in the chlamydial research field. Computer program, which based on the hydro- phobic feature of identified Inc proteins, predicted 90 CPn candidate Inc proteins by autoscreeing open reading frame of Chlamydia pneumonia, but only a few were proved to be in inclusion membrane by antiboby labeling method. Evidences suggest that not all predicted Inc proteins by the computer prediction are localized in the inclusion membrane of chlamydial organism- infected cells and not all Inc proteins can be predicted by this approach. Therefore, there is an urgent need to directly identify/confirm new Inc proteins in the C. pneumoniae-infected cells using chlamydial protein-specific reagents and to further study their biology functions, and it will benefit to deeply understand the Chlamydial biology, pathogenesis and prevention.CPn0308 is one of the predicted Inc proteins,it needs further to experi- mentally confirm whether CPn0308 indeed is localized in the inclusion membrane within infected host cell. Hence, this study is to analysis charcaterics of CPn0308 and its associated genes through CPn gene bank, to clone CPn0308 and its related genes and express them by GST fusion protein model, to make polyclonal antibodies and monoclonal antibodies to these GST fusion proteins, to localize the endogenous proteins by antibody labeling method, and to further characterize the endogenous CPn0308 protein. It will provide importmant information on the potential roles of Inc proteins in C. pneumoniae pathogenesis and prevention.Part one Cloning and expression of CPn0308 and its related genes Objective: To analysis structure feature of CPn0308 and its associated genes with the help of computer prediction, including CT249, MoPn0520 and GPIC0474, and to further clone these genes and express GST fusion proteins, which will provide the basis for the following antibodies preparation and Inc protein identification.Methods:①The information of CPn0308 was retrieved according to reference mentioned. On Basis of the retrieved hints, the relationship among them was further analyzed.②The primers were designed to clone the genes, including CPn0308, CT249, MoPn0520, GPIC0474 and to screen pGEX-6P2. The target genes were obtained by PCR.③The target genes amplified were purified by phenol-chloroform method and further digested by restriction enzymes.④After being purified, the digested PCR products were linked with pre-treated pGEX-6P2 vector via T4 ligase, the recombinants were trans- formed into competent cell(XL1-blue), then cultured on the selected LB plates contained 100μg/ml AMP overnight at 37℃.⑤The positive colonies were selected using PCR directed towards pGEX-6P2 vector, the plasmids were extracted from the positive colonies and further identified using cross-PCR, the positive plasmids were sequenced and analyzed.⑥After the cross-PCR and sequence analysis, the correct colonies were induced with IPTG and the GST fusion proteins were expressed and purified by Glutathione Sepharose TM 4B, then identified with SDS-PAGE gel electrophoresis.Results:①CPn0308 gene with 366bps in length encoded 121 amino acid residues with the molecular weight of 12.971kDa was cloned. CT249, MoPn- 0520 and GPIC0474 which were similar to the CPn0308 in gene structure were also cloned. A gene consisted one cluster alone, was flanked by two known genes, glgP (alpha glycan phosphorylase) and dnaA, the number of the amino acid residues which was encoded by its corresponding gene respectively was similar.②After being digested by the BamHI and NotI, the recombinant pGEX-6P2/CPn0308, pGEX-6P2/CT249, pGEX-6P2/MoPn0520 and pGEX-6P2/GPIC0474 were obtained via directional insertion those digested products into pGEX6P2 vector pre-treated with the same way as that of the purified PCR products. After initial screening of recombinants using primers designed against pGEX6P2, recombinant plasmids were extracted from the positive colonies and confirmed by cross-PCR using one primer from pGEX6P2 and another from target gene. The amplified products were same as the expected ones respectively. The nucleotide sequence of the recombinant plasmid pGEX-6P2/CPn0308, pGEX-6P2/CT249, pGEX-6P2/ MoPn0520 and pGEX-6P2/GPIC0474 was analyzed by DNA sequence analyzer. By blasting those sequences with gene bank, the homology of sequence was 100% for all four cloned genes. All of them possess two transmembrane regions.③The selected positive colonies were induced to express recombinant protein. After being purified, recombinant proteins were identified by SDS-PAGE electrophoresis, the bands and molecular size of those recombined fusion proteins were consistent with the expected ones respectively.Conclusion:①The gene information on CPn0308 and its related genes, including CT249、MoPn0520 and GPIC0474, has been analyzed and they share a similar gene structure.②T he target genes, including CPn0308, CT249, MoPn0520 and GPIC0474, were successfully cloned into pGEX-6P2 vector. The fusion proteins with the glutathione-s-transferase (GST) of CPn0308 and CT249, MoPn0520 and GPIC0474 have been expressed.Part two Preparation of antibodies against four GST fusion proteins and preliminarily applicationObjective: To make polyclonal antibodies(pAbs) to GST-CPn0308 and other three GST fusion proteins and monoclonal antibodies(mAbs) against GST-CPn0308. To localize the four endogenous proteins preliminarily using antibody-labeling method, and to lays the foundation for thoroughly inquiring the biology function of these hypothetical proteins.Methods:①The immunogens were made by purifying GST-CPn0308, GST-CT249, GST-MoPn0520 and GST-GPIC0474 with Glutathione Sepha- rose TM 4B. The purified fusion proteins were used to immunize mice as routine way, the pAbs to four fusion proteins were obtained by separating serum from whole blood. Besides above mentioned, mAb to GST-CPn0308 were also made by the household method.②To screen positive clones and titrate mAbs using indirect immunofluorescence assay (IFA).③The class and subclass of mAbs to CPn0308 were also identified by IFA. The recognition specificity and sites of mAbs were identified with Western Blotting method.④The mouse anti-fusion protein antibodies were used to preliminarily localize the four endogenous proteins in corresponding Chlamydia-infected cells using an IFA. In order to observe the homology between CPn0308 and other three proteins, apart from absorption experiment, antibody to GST-CPn0308 was also used to directly test HeLa cells infected with four different species Chlamydia using IFA.Results:①The sera were titred by IFA as following: CPn0308 was 1:2000, and that of MoPn0520, CT249 and GPIC0474 was 1:200, 1:2000 and 1:200 respectively.②The four hybridoma stains which produced mAbs to CPn0308 were obtained, designed as 2D7、3A6、3H5、5E10. All of them were IgG class. Of all, 2D7 was IgG3, 3A6 was IgG1, both 3H5 and 5E10 were IgG2b. All of them recognized N-terminus of CPn 0308.③IFA indicated that hypothetical protein CPn0308, CT249 and MoPn0520 were in the inclusion membrane,but hypothetical protein GPIC0474 was not localized in inclusion membrane.④Comparing with contrast, no inclusion membrane staining was found if anti-GST-CPn0308 antibodies were pre-absorbed with GST-CPn0308, but inclusion membrane staining could be observed when anti-GST-CPn0308 antibodies were pre-absorbed with GST-CT249, GST-MoPn0520 or GST- GPIC0474. Serum to GST-CPn0308 only recognized inclusion membrane within HeLa cells infected by AR39, but not recognized inclusion membrane within HeLa cells infected by other three species of Chlamydia.Conclusion:①The four hybridomas(2D7, 3A6, 3H5 and 5E10) have been successfully obtained. All are IgG class and recognize N-terminus of CPn0308.②Hypothetical proteins CPn0308, CT249 and MoPn0520, not GPIC0474 have been found to be localized in inclusion membrane with antibody-labeling method.③It has been proved experimentally that CPn0308 has no homologues with CT249, MoPn0520 or GPIC0474. Part three Preliminary study on characteristics of hypothetical protein CPn0308Objectives: To identify hypothetical protein CPn0308 using antibody labeling approach, and further to study its characterization preliminarily.Methods:①Using pAb or mAb to CPn0308 as first antibody, IFA was carried out to observe the localization of the hypothetical protein CPn0308 within infected cells under the fluorescence microscope.②Costaining of CPn0308 with four other proteins from chlamydia pneumonia (CPAFcp、IncA、MOMP and HSP60 as controls ) was processed to observe coloca- lization of CPn0308 with control proteins under the laser confocal microscope.③The recombinants pDsRed-C1/CPn0308 and pDsRed-C1/CPn0186 were transfected into HeLa cells using the lipofectamine 2000 transfection reagent to characterize the staining of the anti-CPn0308 antibodies under the fluorescence microscope. Absorption experiment was done to visualize specificity of the inclusion membrane staining using antibodies to CPn0308 or IncA. The Western Blotting assay was carried out for testing specificity of the anti-CPn0308 antibodies reacted with endogenous and exogenous CPn0308.④The HeLa cells were infected with AR39 organisms for various periods of time, and the culture samples were subjected to IFA to test the expression model of endogenous CPn0308.Results:①The hypothetical protein encoded by C. pneumoniae open reading frame (ORF) of CPn0308 was detected preliminarily in inclusion membrane of C. pneumoniae-infected cells using antibodies raised with CPn0308 fusion proteins.②The anti-CPn0308 mAbs detected a dominant inclusion membrane signal, which was similar to the signal revealed by the anti-IncA, but not the anti-CPAF, anti-MOMP, or anti-HSP60 antibodies. The inclusion membrane localization of CPn0308 was further verified using the laser confocal microscopy. The anti-CPn0308 labeling did not colocalize with CPAFcp, MOMP, or HSP60 (staining showed green, red and blue color), but clearly overlapped with the anti-IncA labeling(staining demonstrated green and yellow color), even at different focal points along the Z axis.③The anti-CPn0308 antibodies only detected the RFP-CPn0308(yellow color)but not the RFP-IncA fusion proteins(red color). The detection of the endogenous antigens in the C. pneumoniae-infected cells by the anti-CPn0308 and anti- IncA antibodies was blocked by the corresponding homologous but not the heterologous GST fusion proteins in an immunofluorescence assay. In the Western Blotting assay, the anti-CPn0308 antibody only recognized a protein band corresponding to the endogenous CPn0308 from the lysates of C. pneumoniae-infected HeLa cells but not HeLa cell alone or C. trachomatis- infected cells.④When the expression of CPn0308 protein was monitored along the infection time, the CPn0308 antigen was detectable 24 hours after infection and remained in the inclusion membrane throughout the infection course.Conclusion:①The first experimental evidence has been provided to directly demonstrate the localization of CPn0308 in the C. pneumoniae inclusion membrane using the anti-CPn0308 pAbs and mAbs.②Coloca -lization demonstrates that distribution pattern of CPn0308 in inclusion membrane is similar to IncA, but not CPAFcp, MOMP and HSP60.③Time course of CPn0308 protein expression has been detected and it has been found that the CPn0308 protein was expressed at 24 h after infection and remained in the inclusion membrane throughout the infection course.Part four Analysis of CPn0308 immunogenicity in the infected patient with CPnObjectives:To analyze CPn0308 immunogenicity in the CPn infected patient with ICVD.Methods:①To collect clinical blood samples and separate plasma from blood and extract the DNA from PBMC.②The CPn IgG and CPn IgM were detected using CPn specified diagnose kit.③CPn DNA from PBMC was assayed using PCR.④Imunogenicity of the endogenous CPn0308 was analyzed by Western Blotting.Results:①Among the 70 patients with ICVD, the positive rate of CPn IgG was 51.43%, and it was higher than that of control; The positive rate of CPn IgM was 2.86%, and that of control was 8.47%.②The positive rate of CPn DNA in PBMC from patients with ICVD was 81.08%,and it was higher than that of control.③After plasma samples were diluted as 1:500, the antibodies to endogenous CPn0308 were still detected using Western Blotting. The reaction was blocked after the plasma was pre-absorbed with GST-CPn0308 beads ahead, but not pre-absorbed with GST-CPn0186 beads.Conclusion:①there is an increase in the positive rate of CPn IgG in the patients with ICVD.②The positive rate of CPn DNA in PBMC from the patients with ICVD is higher than that of the control. The positive rate for CPn by PCR is higher than that of EIA, and CPn DNA detection is a more sensitive method.③The studies preliminarily demonstrate that CPn0308 has immuno- genicity in the individual infected with CPn.更多还原