我国不同利什曼病流行区利什曼原虫分离株核糖体内转录间隔区1的多态性分析

Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China

目的分析我国不同利什曼病流行区的利什曼原虫分离株核糖体内转录间隔区1 (ITS-1)序列的多态性。方法实验室培养10个分离自我国不同利什曼病流行区的利什曼原虫分离株,包括四川/甘肃山丘型内脏利什曼病流行区的SC6和Cy分离株,新疆喀什人源型内脏利什曼病流行区的801和KS-2分离株,新疆伽师荒漠型内脏利什曼病流行区的JIASHI-1、 JIASHI-5、 XJ771分离株以及新疆克拉玛依皮肤利什曼病流行区的KXG-Xu、KXG-Liu和KXG-927分离株。收集利什曼原虫前鞭毛体,提取DNA, PCR扩增各利什曼原虫分离株的ITS-1并送测序,用GENEDOC软件对ITS-1序列进行比对分析,从GenBank下载利什曼原虫其他分离株的ITS-1序列,构建系统进化树,分析我国利什曼原虫分离株的亲缘关系。结果我国10个利什曼原虫分离株均扩增出单一的条带。序列分析结果显示, 801和KS-2分离株的ITS-1片段为316 bp,与MHOM/IN/80/DD8参照株比较其碱基突变率为0; JIASHI-1、 JIASHI-5和XJ771分离株为313 bp,碱基突变率为1.6%; KXG-Xu、 KXG-Liu和KXG-927分离株为312 bp,碱基突变率为1.6%; Cy和SC6分离株为318 bp,碱基突变率为0.6%。系统进化树显示,这10个利什曼原虫分离株ITS-1序列聚集成2个群和3个亚群,其中JIASHI-1、 JIASHI-5和XJ771分离株聚集为A亚群, KXG-Xu、 KXG-Liu和KXG-927分离株聚集为B亚群, A和B亚群聚集于群Ⅰ,为婴儿利什曼原虫; Cy、SC6、 801和KS2分离株聚集为C亚群,属群Ⅱ,为杜氏利什曼原虫。结论我国不同利什曼病流行区的利什曼原虫分离株的ITS-1序列存在多态性。

Objective The polymorphism of the internal transcribed spacer 1 (ITS-1) sequence of Leishmania isolates from different endemic areas in China was analyzed to understand the genetic variation and relationship among different Leishmania isolates.Methods Total 10 Chinese Leishmania isolates,including SC6 and Cy isolates from endemic areas of mountainous visceral leishmaniasis in Sichuan/Gansu Province (Leishmania donovani);801 and KS-2 isolates from endemic areas of anthroponotic visceral leishmaniasis in Kashi,Xinjiang (L.donovani);JIASHI-1,JIASHI-5 and XJ771 isolates from endemic areas of desert-type visceral leishmaniasis in Jiashi,Xinjiang (L.infantum);KXG-Xu,KXG-Liu and KXG-927 isolates from endemic areas of cutaneous leishmaniasis in Karamay,Xinjiang (L.infantum),were maintained in the laboratory culture.The genomic DNAs were extracted from the cultured promastigotes and the ITS-1 was PCR amplified using gene-specific primers.The PCR products of amplified ITS-1 were sequenced and the obtained sequences from different isolates were aligned with reference ITS-1 sequences of Leishmania deposited in GenBank using GENEDOC software.The phylogenetic tree was constructed based on their genetic variation and relationship.Results A single band of ITS-1 with size of about 310 bp was amplified from all 10 Chinese Leishmania isolates.The sequence analysis identified that ITS-1 amplified from anthroponotic L.donovani 801 and KS-2 isolates contained 316 bp with sequence identical to ITS-1 sequence from L.donovani reference strain MHOM/IN/80/DD8 without any mutation.The ITS-1 amplified from desert-type L.infantum JIASHI-1,JIASHI-5 and XJ771 isolates were 313 bp with 1.6% sequence variation.The ITS-1 amplified from cutaneous L.infantum KXG-Xu,KXG-Liu and KXG-927 isolates were 312 bp with 1.6% sequence variation.The ITS-1 amplified from mountainous L.donovani Cy and SC6 isolates were 318 bp with 0.6% sequence variation.Phylogenetic analysis of the ITS-1 sequences showed that the 10 Leishmania isolates were branched into three subgroups and two clusters according to the different species and endemic areas.The L.infantum isolates from Jiashi,Xinjiang (JIASHI-1,JIASHI-5 and XJ771) were closely related and clustered as subgroup A.The L.infantum isolates from Karamay,Xinjiang (KXG-Xu,KXG-Liu and KXG-927) are clustered as subgroup B.Subgroup A and B are closely related to L.infantum reference as cluster Ⅰ.The L.donovani isolates Cy,SC6,801 and KS2 are closely related as subgroup C,fallen into cluster Ⅱof L.donovani.Conclusion ITS-1 sequences of Leishmania isolates from different leishmaniasis endemic areas in China showed different polymorphism related to different Leishmania species and endemic areas.