恶性疟原虫FCC1/HN株己糖激酶基因的扩增、克隆和序列分析(英文)

AMPLIFICATION, CLONING AND SEQUENCE ANALYSISOF THE HEXOKINASE GENE OF PLASMODIUMFALCIPARUM ISOLATE FCC1/HN

目的扩增恶性疟原虫FCC1/HN株的己糖激酶(HK)编码基因,构建其原核和真核表达重组质粒,测定其序列,比较它及它推导的蛋白质与恶性疟原虫其他株和人之间的差异.方法用PCR方法从FCC1/HN株基因组中扩增HK基因;将它分别定向克隆到原核表达质粒pET-30a(+)和真核表达质粒pcDNA3,分别转化大肠埃希菌BL21/DE3 和JM109感受态细菌;经酶切、PCR扩增鉴定筛选到阳性重组克隆,用双脱氧链末端终止法测定其序列,用生物信息学软件分析HK序列及进行同源性比较.结果 PCR扩增得到特异的FCC1/HN株HK基因,大小为1 482 bp,编码493个氨基酸.其氨基酸序列与3D7株完全相同,与K1株的同源性高达99.8%,但与人的4型HK的同源性只有23.2%～26.6%.结论获得恶性疟原虫FCC1/HN株的HK基因,成功构建了其原核和真核表达质粒,并测定了其序列;恶性疟原虫的HK在不同的分离株间高度保守,但与人的同源性很低,可能是一个潜在的药物靶标.

Objective To amplify the hexokinase (HK) gene of Plasmodium falciparum isolate FCC1/HN, compare the sequence with that of other P. falciparum isolates and human, and construct its prokaryotic and eukaryotic expression plasmids. Methods The HK gene was amplified by PCR from the genomic DNA of P. falciparum isolate FCC1/ HN. The amplified HK gene was cloned into the prokaryotic and eukaryotic expression vectors, pET-30a(+) and pcD-NA3 , and transferred into Escherichia coli BL21/DE3 and JM109, respectively. The positive recombinant pET-30a(+)-HK and pcDNA3-HK were screened and identified by endonuclease digestion and PCR. The nucleotide sequence of HK gene was determined by the dideoxy chain termination method. The DNA sequence and its deduced protein sequence were analyzed. Results The HK gene of P. falciparum isolate FCC1/HN was specifically amplified, and the correct recombinant plasmids pET-30a(+)-HK and pcDNA3-HK were constructed. The results of sequencing the nucleotide acids showed that the full length HK gene of P. falciparum isolate FCC1/HN was 1 482 base pair. It encoded a protein of 493 amino acids. The amino acids sequence was the same as that of isolate 3D7, with 99. 8% identity of the isolate K1, but with 23. 2%-26. 6% identity of all the 4 types of the human HK. Conclusion The HK gene of P. falciparum isolate FCC1/HN was amplified. Its recombinant prokaryotic and eukaryotic expression plasmids were successfully constructed and its nucleotide sequence were determined. The HK gene of P. falciparum is quite conserved among different isolates, but has a low homology with human HK. The HK of P. falciparum may be a potential drug target.