摘要
目的测定我国间日疟原虫不同地理株红内期SSUrRNA基因序列,比较分析其分子特征。方法收集深圳、海南、湖北和河南四地间日疟患者血样5份(其中海南2份),并提取核酸DNA,采用PCR从核酸提取物中扩增出间日疟原虫SSUrRNA基因片段,纯化后分别与pGEM-Teasy质粒连接构建重组子并转化大肠杆菌JM109;阳性克隆以双酶切鉴定后,双脱氧末端终止法测定序列,采用BLAST和MEGA4软件分析序列相互关系特征。结果 5株间日疟原虫SSUrRNA基因扩增片段大小一致,约为998bp;核酸序列测定结果显示,所克隆的SSUrRNA基因片段均含有998个核苷酸,不同地理株间SSUrRNA基因序列有9个位点存在变异的可能,两两比对的同源性均高于99.5%,其中河南与湖北株序列的同源性为100.0%;与GenBank中报道的国外6株间日疟原虫相同序列作分子系统进化分析,国内5株间日疟原虫SSUrRNA基因序列与Sal 1株遗传距离小,亲缘关系接近。结论测定的国内间日疟原虫SSUrRNA基因序列在不同地理株间相对保守,其间存在的单核苷酸多态性与地理变化有一定程度的关系。
Aim To clone and analyze the sequence of SSUrRNA-encoding gene （SSUrDNA） fragment from different geographical isolates of P. vivax at blood stage in China. Methods Five infected P. vivax blood samples were collected from Shenzhen City, the provinces of Hainan, Hubei and Henan. The SSUrDNA fragments were amplified by PCR from the DNA extractes of the infected blood samples. After purification, the gene fragments were ligated with plasmid pGEM-Teasy to construct recombinant plasmids,and transformed into E.coli JM109 respectively. Positive bacterial clones were identified by PCR and double enzymes digestion methods. The sequences of inserted SSUrDNA fragments were determined and analyzed by BLAST and MEGA4 software. Results The amplified SSUrDNA fragments were about 998 bp in length,and 9 nucleotide variant sites were found. The results of pair comparison between the different isolate revealed the general homology was above 99.5% ,and the SSUrDNA sequence of Henan isolate was the same as that of Hubei isolate. Based on the SSUrDNA sequence, phylogenetie analysis indicated the isolates in this study were closely associated with Sal 1 strain in genetic relationship. Conclusion The SSUrDNA sequence of P. vivax at blood stage was relatively conserved among different geographical isolates, and the single nucleotide polymophism of the SSUrDNA ,was related with geographical change in some degree.
出处
《中国热带医学》
CAS
2011年第4期401-403,共3页
China Tropical Medicine
基金
深圳市科技计划资助项目(No.200902090)
作者简介
高世同（1969～），男，硕士，主任医师，主要从事感染性疾病研究。通讯作者：E-mail：gst@szcdc．net
