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目的分析我国婴儿利什曼原虫汶川株动基体小环DNA株内序列差异性。方法应用来自小环DNA保守区的一对引物KP1-KP2扩增汶川株婴儿利什曼原虫动基体小环DNA。扩增产物克隆于pGEM-T载体,随机选取20个阳性克隆进行双向测序,应用DNAStar软件进行比对分析和开放阅读框预测,利用Mega5.0构建系统进化树。结果应用KP1-KP2引物从汶川株婴儿利什曼原虫扩增出动基体小环DNA片段,长约850bp。经序列比对可将20个阳性克隆序列区分为CQ18和CQ07两大类,进化分析显示这两类序列与杜氏利什曼原虫种团的原虫亲缘关系最近。CQ07序列含一较大开放阅读框,预计编码69个氨基酸残基的蛋白。结论婴儿利什曼原虫汶川株动基体小环DNA株内序列存在差异。
OBJECTIVE: To analyze the DNA sequence variation of the chimeric small loop of Leishmania infantum Wenchuan strain in China. Methods A pair of primers KP1-KP2 from the conserved region of small loop DNA was used to amplify the DNA of the chorionic circulation of Leishmania infantum. The amplified product was cloned into pGEM-T vector and 20 positive clones were randomly selected for bidirectional sequencing. DNAStar software was used for comparison analysis and open reading frame prediction. Mega5.0 was used to construct phylogenetic tree. Results The KP1-KP2 primer was used to amplify the small segment DNA fragment of Leishmania infantum, which was about 850 bp in length. Twenty positive cloned sequences could be divided into two major categories CQ18 and CQ07 by sequence alignment. Phylogenetic analysis showed that the two sequences were closest to the protozoan Leishmania donovani. The CQ07 sequence contains a large open reading frame and is expected to encode a protein of 69 amino acid residues. Conclusion There are differences in DNA sequence of the chymotrypsin of Leishmania infantum Wenchuan strain.