我国不同流行区利什曼原虫分离株动基体小环DNA序列分析

Sequence analysis on kinetoplast DNA minicircles of Chinese Leishmaniaisolates in different foci

目的分析我国不同流行区利什曼原虫动基体小环DNA序列差异性。方法应用来自小环DNA保守区的一对引物KP1-KP2扩增该利什曼原虫动基体小环DNA,将扩增产物克隆于pGEM-T载体,随机选取5个阳性克隆进行双向测序,对获得的小环序列应用DNAStar软件进行比对分析,并利用Mega5.0构建系统进化树。结果应用KP1-KP2引物分别从来自我国不同流行区利什曼原虫虫株SC、KXG-Xu、KXG-65、JIASHI-1和801扩增出单一小环序列,长度分别为858bp,858bp,858bp,750bp和748bp。SC、KXG-Xu和KXG-65间序列一致性超过99%,JIASHI-1和801虫株序列均与其它虫株显示高度异质性。进化分析显示801与一杜氏利什曼原虫聚为一类,JIASHI-1与亲内脏的利什曼原虫聚为一大类,而SC、KXG-Xu和KXG-65三者聚为独自一类,和亲皮肤的利什曼原虫相比,与亲内脏的利什曼原虫有更近的亲缘关系。结论我国不同流行区利什曼原虫虫株小环序列存在较大差异性。

This study conducted an analysis for the sequence heterogeneity of the kinetoplast DNA minicircles in Chinese Leishrnania isolates in different foci. Kinetoplast DNA minicircles were amplified using a set of primers from conserved region of minieircle of Leishrnania. The PCR products were then cloned into pGEMT vector and 5 positive clones were selected to be sequenced randomly. Sequence alignment was done by DNAStar software and phylogenic tree was constructed using MEGA software. Results revealed that single minicircles were amplified from Leishmania strains SC, KXG-Xu, KXG-65, JIASHI-1, and 801 from different foci of China using primer KP1-KP2, with the length of 858 bp, 858 bp, 858 bp, 750 bp, and 748 bp, respectively. The sequence identity among SC, KXC-Xu and KXG-65 were over 99 %, and JIASHI-1 and 801 both showed high heterogeneity with other strains of Leishmania. Strain 801 was close to the isolates of Leishmania donovani, and the strain JIASHI-1 was close to the isolate of viscerotropic Leishmania. SC, KXG-Xu and KXG-65 were also clustered together, while they were closer to viscerotropic Leishmania than to cutaneotropic Leishmania. It showed that sequence variations exist in ki- netoplast minicircles of Leishmania from different loci of China.