恶性疟原虫FCC1/HN株丙酮酸激酶基因的扩增、克隆和序列分析

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。

AMPLIFICATION, CLONING AND SEQUENCE ANALYSIS OF THE PUTATIVE PYRUVATE KINASE GENE OF PLASMODIUM FALCIPARUM(FCC1/HN)

Objective To amplify the pyruvate kinase (PK) gene of Plasmodium falciparum isolate FCC1/HN, compare the sequence with the corresponsing genes of P. falciparum 3D7 and other organisms, and construct its prokaryotic and eukaryotic expression plasmids. Methods A pair of primers was designed according to the putative PK gene of P. falciparum 3D7. The PK gene was PCR amplified from the genomic DNA of P. falciparum isolate FCC1/HN. The amplified PK gene was cloned into the prokaryotic and eukaryotic expression vectors, PGEX-4T-1 and pcDNA_ 3 , and transformed into E. coli JM109 respectively. The positive recombinant PGEX-4T-1-PK and pcDNA_ 3 -PK were screened and identified by endonuclease digestion and PCR. The nucleotide sequence of PK gene was determined by the dideoxy chain termination method. The DNA sequence and the deduced protein sequence were analyzed. Results The PK gene of isolate FCC1/HN was specifically amplified, and the recombinant plasmids PGEX-4T-1-PK and pcDNA_ 3 -PK were constructed. The results of DNA sequencing showed that the full length PK gene of isolate FCC1/HN was 2 235 bp. It was encoded a protein of 744 amino acids. The amino acids sequence showed 99.9% and 70.2% identities with that of the isolate 3D7 and P. yoelii yoelii respectively, while with 20.9% and 21.6%-25.4% identities of that of Toxoplasma gondii and all types of the human PK respectively. Conclusions The PK gene of P. falciparum isolate FCC1/HN was amplified from genomic DNA. Its recombinant prokaryotic and eukaryotic expression plasmids were successfully constructed and its nucleotide sequence was determined. The DNA sequence was quite conserved with 3D7, but showed a low homology with human PK genes. The PK of P. falciparum may be a potential drug target.