Molecular Cloning, Characterization, and Expression of the M Antigen of Histoplasma capsulatum

The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.

荚膜组织胞浆菌(Histoplasma capsulatum)的主要诊断抗原为H抗原与M抗原，二者均为多效能糖蛋白，可同时诱导体液免疫与T细胞介导的免疫应答。这类抗原可能参与组织胞浆菌病的致病过程。其中M抗原被认为是免疫显性抗原，因为针对该抗原的抗体是急性组织胞浆菌病中最早出现的沉淀素，且在感染的所有阶段均普遍存在。目前对于单分子M抗原的生物学活性及其诱导针对荚膜组织胞浆菌的保护性免疫应答的能力，尚不完全明确。本研究采用分子生物学方法解析M抗原的生物学特性，包括从组织胞浆菌素(histoplasmin)中纯化该抗原、使用蛋白酶进行部分酶解，以及通过反相高效液相色谱(Reverse-phase high-performance liquid chromatography)分离酶解释放的肽段。通过埃德曼降解(Edman degradation)获取纯化肽段的氨基酸序列，并利用简并寡核苷酸引物进行聚合酶链式反应(PCR)，从荚膜组织胞浆菌的基因组DNA中扩增得到一段321 bp的M抗原编码基因片段。利用该片段筛选荚膜组织胞浆菌基因组DNA文库，最终获得并克隆了全长基因并完成测序。M基因全长2187 bp，编码分子量为80719 Da的蛋白质，该蛋白与烟曲霉(Aspergillus fumigatus)、黑曲霉(Aspergillus niger)以及构巢曲霉(Emericella nidulans)的过氧化氢酶具有显著同源性。通过逆转录聚合酶链式反应(Reverse transcription-PCR)获取互补DNA(cDNA)，并将其克隆至表达载体pQE40中。通过蛋白质印迹法(Western blotting)验证了克隆表达的蛋白的身份：该重组融合蛋白可与针对M抗原的单克隆抗体、小鼠抗M抗原多克隆抗血清，以及一名组织胞浆菌病患者的血清样本发生免疫反应。本研究表明，编码荚膜组织胞浆菌主要免疫显性M抗原的基因属于推定的过氧化氢酶基因，且重组蛋白保留了血清学诊断活性。