论文部分内容阅读
目的 扩增恶性疟原虫海南株FCC1/HN的丙酮酸激酶 (PK)的编码基因 ,构建其原核和真核表达重组质粒 ,测定其序列 ,并了解它及它推导的蛋白质与恶性疟原虫 3D7和人PK基因之间的差异。 方法 根据 3D7的PK基因设计合成一对引物 ,用PCR从FCC1/HN株基因组中扩增PK基因 ;将它分别定向克隆到原核表达质粒PGEX 4T 1和真核表达质粒 pcDNA3 ,分别转化大肠杆菌JM 10 9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PK序列及进行同源性比较。 结果 PCR扩增得到特异的FCC1/HN株PK基因 ,大小为 2 2 3 5bp ,编码 744个氨基酸。其氨基酸序列与 3D7的同源性高达 99.9% ,与约氏疟原虫的同源性为 70 .2 % ,但与弓形虫和人的PK的同源性分别只有 2 0 .9%和 2 1.6%～ 2 5 .4%。 结论 从FCC1/HN株基因组中获取了PK基因 ,成功构建了其原核和真核表达质粒 ,并测定了其序列 ;它与 3D7的PK高度同源 ,但与人的PK基因同源性很低 ,可能是一个药物靶标。
Objective To amplify the gene encoding pyruvate kinase (PK) of Plasmodium falciparum Hainan strain FCC1 / HN, construct its prokaryotic and eukaryotic recombinant plasmids, determine its sequence, and understand its relationship with its deduced protein and Plasmodium falciparum 3D7 and Differences between human PK genes. Methods According to the PK gene of 3D7, a pair of primers was designed and synthesized. The PK gene was amplified from the genome of FCC1 / HN strain by PCR. The PK gene was cloned into prokaryotic expression plasmid PGEX 4T 1 and eukaryotic expression plasmid pcDNA3 respectively and transformed into E. coli JM 10 9 Competent bacteria. The positive recombinant clones were screened by restriction enzyme digestion and PCR amplification. The sequences were determined by dideoxy chain termination method. The PK sequences were analyzed by bioinformatics software and the homology was compared. Results The PK gene of FCC1 / HN strain was amplified by PCR and its size was 2 2 3 5 bp, which encoded 744 amino acids. Its amino acid sequence is 99.9% homologous to 3D7 and 70.2% homologous to Plasmodium yoelii, but its homology with Toxoplasma gondii and human PK is only 20.9% and 2 1.6, respectively % ~ 2 5 .4%. Conclusion The PK gene was obtained from the genome of FCC1 / HN strain and its prokaryotic and eukaryotic expression plasmids were successfully constructed and sequenced. It is highly homologous to the PK of 3D7 but has low homology with human PK gene , May be a drug target.