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

原生动物寄生虫利什曼原虫含有多个P-糖蛋白基因，是一个多基因家族。从不同种类的利什曼原虫中分离到两种类型的基因(Ldmdrl和LtpgpA)，并发现它们具有抗药性。在这项研究中，我们旨在分离和鉴定新大陆皮肤利什曼病的病原体之一--亚马孙链霉菌的P-糖蛋白基因。利用针对哺乳动物P-糖蛋白和利什曼原虫P-糖蛋白基因保守序列的特异引物，通过聚合酶链式反应(PCR)扩增亚马孙钩端螺旋体P-糖蛋白中的ATP结合位点。扩增产物大小为408个碱基，在氨基酸水平上与Ldmdrl和LtpgpA的同源性分别为68%和37%，提示该片段属于新型利什曼原虫P-糖蛋白基因。为了获得完整的编码基因，构建了亚马孙链霉菌基因组文库，并将其克隆到lambda-gem-11载体中，对扩增产物进行筛选。将其中一个阳性克隆用Sac I酶切后，将DNA片段亚克隆至pUC19载体。目前，一个1005bp的Sac I片段的核苷酸测序已经完成，其余片段已准备好进行测序。此外，脉冲场凝胶电泳法和Southern杂交分析有力地表明，亚马孙龙虾至少含有三种不同类型的P-糖蛋白基因，即Ldmdrl同源物、LtpgpA同源物和本研究中发现的新型基因，但LtpgpA与其他两个基因在染色体上的位置不同。

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.