Molecular Biological Studies on the Proteases and .Their Inhibifors of Filarial Parasites

从马来丝虫cDNA文库中克隆了两个天冬氨酸蛋白酶的全长cDNA和一个天冬氨酸蛋白酶的部分cDNA，测定了它们的碱基序列，并推导了它们的氨基酸序列。系统进化树分析表明，马来丝虫的两种主要天冬氨酸蛋白酶属于不同的天冬氨酸蛋白酶类群。使用其抗体进行的组织化学分析表明，其中一种仅存在于食道和肠道中，而另一种存在于各种组织和器官中，包括食道，肠道，体壁和生殖器官。这些结果表明，前者参与营养蛋白的消化，而另一个负责细胞内蛋白质的降解。三个全长cDNA。另外，还克隆了4个半胱氨酸蛋白酶的部分cDNA，1个金属蛋白酶的全长cDNA和4个部分cDNA，以及2个去泛素化酶的cDNA。 关于我们 分离纯化，并测定其序列以推断相应的氨基酸序列。一种金属蛋白酶的全长cDNA，其编码序列与线粒体加工蛋白酶的非催化亚基相似。尝试了使用大肠杆菌和短芽孢杆菌表达系统异源表达这些蛋白酶中的一些。在急诊大肠杆菌系统中，产生了相当数量的蛋白酶，但为无活性的包涵体。然而，尽管在不同条件下进行了许多尝试，但变性酶的重折叠成潜在活性酶是相当困难的。B。Brebis系统也未能给出合理量的潜在活性酶。在这方面需要进行进一步的详细研究。沿着这些研究，我们利用cDNA克隆和RNA干扰技术对秀丽隐杆线虫（一种非寄生线虫）的几种蛋白酶进行了比较研究，并鉴定了几种重要的蛋白酶。少

Full-length cDNAs of two aspartic proteases and a partial-length CDNA of one aspartic protease were isolated from the CDNA libraries of Brugia malayi and their base sequences were determined and amino acid sequences deduced. Analysis of a phylogenetic tree indicated that the two major aspartic proteteases from Brugia malayi belong to different groups ofaspartic proteases. Histochemical analysis using their antibodies showed that one of them is present only in the esophagus and intestines while the other is present in various tissues and Organs including the esophagus, intestines, body wall and reproductive organs. These results suggested that the former is involved in the gastro-intestinal digestion of nutrient proteins while the other is responsible for ihtracellular protein degradation. Three full-length cDNAs. and four partial-length cDNDs of cysteine proteases, one full-length CDNA and four partial-length cDNAs of metalloproteases, and two cDNAs of deubiquitinating enzymes were als … More o isolated, and their sequences were determined to deduce the corresponding amino acid sequences. The full-length CDNA for a metalloprotease coded for a sequence sinmilar to that of the non-catalytic subunit of mitochondrial processing protease. Heterologous expression of some of these proteases were, attempted using Escherichial coli and Bacillus brevis expression systems. In the E. coli system, aconsiderable amount of the proteases was produced, but as an inactive inclusion bodies. The refolding of the denatured enzymes to potentially active enzymes, however, was found to be rather difficult, despites of a number of attempts under different conditions. The B. brebis system also-failed-to give a reasonable amount of the potentially active enzymes. Further elaborate studies are necessary in this regard. Along with these studies, a comparative studies were carried out with several proteases Caenorhabditis elegans, a non-parasitic nematode, using CDNA cloning and RNA interference, and several important proteases were identified. Less