Kohlstädt S, Couissinier-Paris P, Bourgois A, Bouchon B, Piper K, Kolbe H, Dessein A J
INSERM U399/Laboratoire de Parasitologie-Mycologie, Faculté de Médecine, Marseille, France.
Mol Biochem Parasitol. 1997 Feb;84(2):155-65. doi: 10.1016/s0166-6851(96)02787-9.
Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.
作为强效T细胞免疫原的寄生虫抗原是针对易受T细胞介导免疫影响的病原体的潜在疫苗候选物。我们之前已经表明，血吸虫提取物的色谱级分含有在人类自然血吸虫感染中作为主要T细胞免疫原的成分，并且可能有助于诱导人类针对这种寄生虫的保护性免疫。在本研究中，我们报告了这些级分的活性成分的分子克隆及生化特性。用针对这些级分产生的抗体筛选血吸虫cDNA表达文库，使得一个与血吸虫幼虫和成虫的0.56-kb mRNA杂交的cDNA得以克隆。该cDNA包含一个267个碱基对（bp）的开放阅读框，其编码一个10-kDa的多肽。对该cDNA序列的分析显示与先前报道的功能未知的蛋白质序列有70%的同源性。在通过反相高效液相色谱（HPLC）进行进一步纯化后，通过质谱对活性级分中的天然分子进行分析。该过程在级分中揭示了分子量为10383 +/- 2 Da和10401 +/- 9 Da的两种成分。这两种多肽均刺激免疫T细胞，并产生其序列与克隆分子的序列匹配的胰蛋白酶肽。这两种多肽可能对应于由克隆的cDNA编码的多肽的不同翻译后修饰形式。
