Mevelec M N, Chardès T, Mercereau-Puijalon O, Bourguin I, Achbarou A, Dubremetz J F, Bout D
Unité de Recherche Université-INRA d'Immunologie Parasitaire, Institut National de la Recherche Agronomique, Nouzilly, France.
Mol Biochem Parasitol. 1992 Dec;56(2):227-38. doi: 10.1016/0166-6851(92)90172-g.
Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.
从弓形虫表达文库中挑选出的克隆，用来自感染弓形虫的兔子的免疫血清进行筛选，然后用经口服感染弓形虫包囊的小鼠的乳汁和肠道分泌物进一步筛选。几个克隆的基因产物与乳汁IgA反应强烈，与肠道IgA反应较弱。其中三个克隆（5.1、36.1、37.4）被证明编码一种40 kDa的致密颗粒蛋白（GRA4）。GRA4蛋白与黏膜IgA识别的一种弓形虫抗原共迁移。通过克隆含有37.4 DNA插入片段的弓形虫基因组BamHI片段，获得了GRA4的完整核苷酸序列。编码序列不含内含子。推导的氨基酸序列表明其产物富含脯氨酸（12%），有一个19个氨基酸的内部疏水区域和一个潜在的N-糖基化位点。理论大小为36260 Da的初级翻译产物含有一个20个氨基酸的推定N端信号序列，但没有明显的糖脂锚定序列。GRA4基因的定量和Southern印迹分析表明GRA4基因是单拷贝的。GRA4基因在速殖子中翻译产生一种约1900个碱基的单一mRNA。
