Mechanisms of drug-resistance and analysis of drug-resistant genes in Leishmania

利什曼原虫耐药机制及耐药基因分析

基本信息

批准号：
05670243
负责人：
KATAKURA Ken
金额：
$ 1.34万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

A protozoan parasite, Leishmania, contains several P-glycoprotein genes as a multigene family. Two types of the gene (Ldmdrl and LtpgpA) have been isolated from different Leishmania species, and found to play a role of drug-resistance. In this study, we aimed to isolate and characterize P-glycoprotein genes in L.amazonensis, one of the causative agents of cutaneous leishmaniasis in the New World. Polymerase chain reaction (PCR) was performed to amplify ATP binding sites within P-glycoproteins of L.amazonensis by using primers specific to conserved sequences in mammalian and leishmanial P-glycoprotein genes. We detected a 408-bp PCR product which showed 68% and 37% identity to Ldmdrl and LtpgpA,respectively, at the amino acid level, suggesting that this DNA fragment is in part of new type of leishmanial P-glycoprotein gene. To isolate the putative gene in whole, a genomic library of L.amazonensis DNA was constructed into the lambda-GEM-11 vector and screened with the PCR product. One of the positive lambda clones was digested with Sac I restriction endonuclease, and resultant DNA fragments were subcloned into pUC19 vector. At present, nucleotide sequencing of a 1005-bp Sac I fragment was completed, and the rest of the fragments is ready to be sequenced. In addition, pulsed field gel electrophoresis and Southern hybridization analysis strongly suggest that the L.amazonensis contains at least three different types of P-glycoprotein gene, the Ldmdrl homologue, LtpgpA homologue and new type gene found in this study, but that chromosomal location of the LtpgpA is different from those of the other two genes.

原生动物寄生虫利什曼原虫（Leishmania）含有几种P-糖蛋白基因作为多基因家族。已经从不同的利什曼原虫物种中分离出两种类型的基因（LDMDRL和LTPGPA），并发现具有抗药性的作用。在这项研究中，我们旨在隔离和表征L.Amazonensis的P-糖蛋白基因，L.Amazonensis是新世界皮肤利什曼病的病因之一。通过使用特定于哺乳动物和利什曼语P-糖蛋白基因的保守序列的引物，通过使用特定于保守序列的L. amazonensis的P-糖蛋白在L.amazonensis的P-糖蛋白中扩增聚合酶链反应（PCR）。我们在氨基酸水平上分别检测到了408 bp PCR产品，该产品分别显示出对LDMDRL和LTPGPA的68％和37％的身份，这表明该DNA片段是新型利什曼尼尔P-糖蛋白基因的一部分。为了整体分离推定的基因，将L.amazonensis DNA的基因组文库构建到Lambda-GEM-11载体中，并用PCR产物进行筛选。用SAC I限制性核酸内切酶消化了一个正Lambda克隆，并将结果的DNA片段亚克隆到PUC19载体中。目前，完成了1005 bp SAC I片段的核苷酸测序，其余片段就可以测序了。此外，脉冲场凝胶电泳和南部杂交分析强烈表明，L.Amazonensis包含至少三种不同类型的P-糖蛋白基因，LDMDRL同源物，LTPGPA同源物和新型基因，但在这项研究中发现了新型基因，但是与LTPGPA的那个染色体位置相关，这些基因与这些基因相同。