Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica
Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica
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DOI：
10.1016/j.molbiopara.2004.02.010
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发表时间：
2004-07-01
影响因子：
1.5
通讯作者：
Sobhon, P
中科院分区：
文献类型：
--
作者：
Meemon, K;Grams, R;Sobhon, P
从成虫、新囊幼虫（NEJ）和包膜期克隆了巨片吸虫编码组织蛋白酶B蛋白酶基因的转录产物。获得的cdna分别命名为FG cat-B1、FG cat-B2和FG cat-B3。所编码蛋白酶的氨基酸序列具有64 ~ 79%的同源性。与同源蛋白的序列比较表明，所有先前描述的组织蛋白酶B的功能重要残基都是保守的。Southern的分析证实了E gigantica基因组中存在一个相关的组织蛋白酶B基因家族。Northern分析显示，在包膜期和NEJ期检测到1400个核苷酸的组织蛋白酶B转录物。组织蛋白酶B转录本通过RNA原位杂交在盲肠上皮细胞、消化道近端细胞和表面被盖细胞中定位。此外，在生殖系统的组织中，包括前列腺细胞、梅氏细胞、卵黄腺、睾丸和卵子细胞中检测到转录本。利用基因特异性引物和标记的组织蛋白酶B探针杂交，采用RT-PCR方法研究了阶段特异性基因表达。在所有阶段均检测到FG cat-B1转录本，而FG cat-B2和FG cat-B3转录本仅在囊蚴、NEJ和幼年寄生虫中表达。在寄生虫成熟过程中，猫- b2和猫- b3基因的关闭意味着这些酶可能在渗透和迁移到肝脏的过程中参与消化宿主组织，而猫- b1在所有阶段都存在，可能执行一般消化功能。(C) 2004年Elsevier B.V.出版
The transcriptional products of Fasciola gigantica genes encoding cathepsin B proteases were cloned from adult, newly excysted juvenile (NEJ), and metacercarial stages. The obtained cDNAs were named FG cat-B1, FG cat-B2, and FG cat-B3. The deduced amino acid sequences of the encoded proteases have identities ranging from 64 to 79%. Sequence comparison with homologous proteins showed that all functional important residues formerly described for cathepsin B are conserved. Southern analysis confirmed the presence of a family of related cathepsin B genes in the genome of E gigantica. Northern analysis revealed a common transcript size of 1400 nucleotides with abundant cathepsin B transcripts detected in metacercarial and NEJ stages. Cathepsin B transcripts were located by RNA in situ hybridization in the caecal epithelial cells, in cells underlining the proximal part of the digestive tract, and in the tegumental cells underlining the surface tegument. Furthermore, transcripts were detected in the tissues of the reproductive system including cells of prostate, Mehlis, and vitelline glands, testis, and eggs. Stage-specific gene expression was investigated by RT-PCR using gene-specific primers and hybridization with a labeled cathepsin B probe. FG cat-B1 transcripts were detected in all stages, whereas FG cat-B2 and FG cat-B3 transcripts were expressed in metacercariae, NEJ, and juvenile parasites only. The switching off of the cat-B2 and cat-B3 genes during the maturation of the parasites implicates that these enzymes may be involved in digesting host tissues during penetration and migration to the liver, whereas cat-B1 present in all stages may perform general digestive function. (C) 2004 Published by Elsevier B.V.