Molecular Biology of Pneumocystis Carinii and its Clinical Application

卡氏肺孢子虫是一种机会性病原体，由于艾滋病、癌症化疗或器官移植免疫抑制治疗，在免疫抑制或免疫缺陷的情况下，经常会导致致命的肺炎。卡氏肺孢子虫是一种真核微生物，可感染多种哺乳动物宿主。卡氏肺孢子虫的连续体外培养尚未建立，因此大规模培养的困难阻碍了对该生物体的研究进展。然而，有必要寻求卡氏肺孢子虫研究的分子方法1。表面糖蛋白。卡氏肺孢子虫的主要表面免疫决定因素是一组称为P115的蛋白质。它们由六个主要的等电变异体组成，并与甘露糖高度糖基化。抗Pl15单抗的频繁产生意味着这些分子具有很强的抗原性。脱糖分析表明，P115的糖基在其抗原性中起着关键作用。PI 15…更多的cdna克隆。用lambdagtll载体构建了卡氏肺孢子虫的cDNA文库。用抗P115蛋白部分的抗体筛选文库，得到40个阳性克隆。核苷酸序列分析表明，它们至少被归类为三个相似但不相同的组。它们与单抗具有相同的表位位置。此外，针对其中一种cDNALacZ融合蛋白的兔多克隆抗体可与完整的P115分子反应。因此，我们确信我们成功地克隆了P115基因。观察到的cdna结构的异质性可能反映了卡氏肺吸虫生物的异质性，或者像在其他寄生原生动物中发现的那样，表面结构的变异性。聚合酶链式反应诊断。我们已经成功地利用聚合酶链式反应(PCR)方法，通过在各种临床和动物样本中扩增5S rDNA序列来检测卡氏肺孢子虫。该检测具有高度的敏感性和特异性。广泛的试验表明，在肺炎患者的痰样本中，PCR方法是有效的。然后，对聚合酶链式反应阳性的患者每天服用扑热息痛或磺胺甲恶唑/甲氧苄啶，并通过聚合酶链式反应检测进行监测。这些试验证明了应用聚合酶链式反应监测卡氏肺炎杆菌肺炎治疗的有效性。_更少

Pneumocystis carinii is an opportunistic pathogen which often causes fatal pneumonia in patients under immunosuppressed or immune deficient conditions due to AIDS, cancer chemotherapy or immunosuppressive therapy for organ transplantation. P. carinii is a eukaryotic microorganism and infects many mammalian hosts. Continuous in vitro culture of P. carinii has not been established and thereby the difficulty of mass cultivation impedes progress in the study of this organism. Nonetheless, it is necessary to pursue molecular approaches to P. carinii studies.1. Surface Glycoproteins. The major surface immunodeterminants of P. carinii is a group of proteins called P115. They are composed of six major isoelectric variants and highly glycosylated with mannose. The frequent generation of anti-Pll5 monoclonal antibodies implicates a strong antigenic activity of these molecules. Deglycosylation analysis revealed that the sugar moiety of P115 plays a crucial role in the antigenic activity.2. PI 15 … More cDNA Cloning. P. carinii cDNA library was constructed using lambdagtll vector. The library was screened with antibodies against the P115 protein moiety, and 40 positive clones were isolated. Nucleotide sequence analyses revealed that they were classified to at least three similar but not identical groups. They share the same epitope site to monoclonal antibodies. Furthermore, rabbit polyclonal antibodies raised against one of these cDNA-lacZ fusion proteins reacted with the intact P115 molecules. Therefore, we were convinced of our cloning of P115 cDNA. The observed heterogeneity in the cDNA structure might reflect heterogeneity in P. carinii organisms or variability of the surface structure as found in other parasitic protozoa.3. Diagnosis with Polymerase Chain Reaction. We have successfully utilized the polymerase chain reaction (PCR) method to detect P. carinii organisms by amplifying the 5S RDNA sequence in various clinical and animal samples. The detection was highly sensitive and specific. Extensive trials manifested efficacy of the PCR method in sputum samples from patients who suffered from pneumonia. The PCR-positive patients were then administered daily with pentamidine or sulfamethoxazole/trimethoprim and were monitored by the PCR assay. These trials demonstrated the efficacy of applying PCR to the monitoring of therapy to P. carinii pneumonia. _ Less