恶性疟原虫海南株子孢子期SSU rRNA编码基因的克隆及序列分析

Cloning and sequencing sporozoite stage SSU rRNA-encoding gene of Plasmodium falciparum Hainan strain

目的克隆恶性疟原虫海南株子孢子期小亚基核糖体核糖核酸（SSUrRNA）编码基因片段，分析其序列特征。方法根据恶性疟原虫基因库相关核酸序列设计1对引物，采用PCR方法从海南恶性疟患者血样核酸提取物中扩增出恶性疟原虫SSUrRNA基因片段，纯化后与pGEM-Teasy质粒连接，构建重组子并转化大肠杆菌JM109；阳性克隆经双酶切鉴定后，双脱氧末端终止法测定序列，采用BLAST软件分析其特征。结果恶性疟原虫子孢子期SSUrRNA基因扩增片段大小约为347bp；阳性克隆重组质粒双酶切及PCR扩增均得到预期大小的片段；核酸序列测定显示插入的SSUrRNA基因扩增片段含有347个核苷酸，与GenBank中的恶性疟原虫3D7株相同序列进行比对，其同源性为100％，而与7G8株的同源性则为98．0％，其中第153位碱基发生了缺失，第184位碱基由C取代了T，而第188位T碱基与第189位A碱基为插入碱基，第243位碱基则由T取代了c。结论成功克隆恶性疟原虫海南株孢子期SSUrRNA编码基因序列，该序列相对保守，不同地理株间存在单核苷酸多态性。

Objective To clone and analyze the specific sequence of sporozoite stage SSU rRNA-enco- ding gene fragment of P. falciparum isolates from Hainan Province in China. Methods The SSU rRNA fragment was amplified by PCR from the DNA extracts of blood sample from a patient with P. falciparum infection in Hainan Province. After purification, the gene fragment was ligated with plasmid pGEM-Teasy to construct a recombinant plasmid, which was transformed into E. coli JM109. Positive bacteria clones were identified by PCR and double enzymes digestion methods. The sequence of inserted SSU rRNA fragment was then determined and analyzed. Results The amplified SSU rRNA fragment was about 347 bp in length, as aligned with the P. falciparum corresponding sequences of 3D7 and 7G8 strains deposited in the GenBank database, the identity of nucleotides was 100% and 98.0% , respectively. Conclusion The P. falciparum sporozoite stage SSU rRNA gene fragment sequence was cloned, which was relatively conserved among different P. falciparum isolates.