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

利什曼原虫是一种含有多个P-糖蛋白基因的多基因家族。两种类型的基因（Ldmdrl和LtpgpA）已从不同的利什曼原虫物种中分离出来，并发现发挥耐药性的作用。在这项研究中，我们的目的是分离和表征的P-糖蛋白基因的亚马逊军团菌，在新大陆的皮肤利什曼病的病原体之一。利用哺乳动物和利什曼原虫P-糖蛋白基因保守序列特异性引物，通过聚合酶链反应（PCR）扩增亚马逊乳杆菌P-糖蛋白的ATP结合位点。结果表明，该片段与Ldmdr 1和LtpgpA的氨基酸序列同源性分别为68%和37%，表明该片段是新型利什曼原虫P-糖蛋白基因的一部分。为了从整体上分离出该基因，将亚马逊乳杆菌DNA的基因组文库构建到Adhesida-GEM-11载体中，并用PCR产物进行筛选。用Sac Ⅰ限制性内切酶消化其中一个阳性λ克隆，将所得DNA片段亚克隆到pUC 19载体中。目前，已完成了一个1005 bp的Sac I片段的核苷酸测序，其余片段已准备好测序。此外，脉冲场凝胶电泳和Southern杂交分析表明，亚马逊乳杆菌中至少含有3种不同类型的P-糖蛋白基因，即Ldmdr 1同源基因、LtpgpA同源基因和本研究发现的新类型基因，但LtpgpA基因的染色体定位与其它两个基因不同。

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.