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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1482 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) coding gene of Plasmodium falciparum FCC1 / HN strain and construct its prokaryotic and eukaryotic recombinant plasmids. The sequence of the recombinant plasmids was analyzed and compared with that of other plasmids and plasmids of Plasmodium falciparum The difference between. Methods The HK gene was amplified from the genome of FCC1 / HN strain by PCR and cloned into the prokaryotic expression plasmid pET-30a (+) and the eukaryotic expression plasmid pcDNA3 respectively and transformed into E. coli BL21 / DE3 and JM109 State bacterium. The positive recombinant clones were screened by restriction enzyme digestion and PCR amplification. The sequences were determined by dideoxy chain termination method. The homology was analyzed by bioinformatics software. Results The genomic DNA of FCC1 / HN strain was obtained by PCR. The size of the HK gene was 1482 bp, encoding 493 amino acids. Its amino acid sequence is exactly the same as that of 3D7 strain, with homology of 99.8% with K1 strain, but only 23.2% ~ 26.6% with human type 4 HK strain. CONCLUSION: The HK gene of Plasmodium falciparum FCC1 / HN strain was obtained and its prokaryotic and eukaryotic expression plasmids were successfully constructed. The sequence of the gene was also determined. HK of Plasmodium falciparum was highly conserved among different isolates but not homologous to human Low sex may be a potential drug target.