恶性疟原虫海南株FEN-1基因的克隆及序列分析

目的　克隆并测定恶性疟原虫海南株(FCC1/HN)侧翼核酸内切酶 1(FEN 1)基因序列,比较 FCC1/HN株与国外分离株FEN 1基因序列的差异。　方法　根据 FEN 1 基因已知序列设计合成 1 对引物,应用 PCR技术从 FCC1/HN株基因组DNA中扩增出FEN 1基因,构建重组质粒PMD18 T FEN 1。阳性克隆的重组质粒经酶切鉴定后进行测序。应用DNAMAN分析软件进行不同分离株FEN 1基因序列的同源性比较。　结果　PCR扩增得到特异的 FCC1/HN株FEN 1基因序列,基因全长1 947 bp,A+T含量为56%,G+C含量为44%。酶切鉴定获得了正确的PMD18 T FEN 1重组质粒。测序表明,恶性疟原虫FCC1/HN株与国外Gambia株和3D7株的FEN 1基因序列有较高的同源性。结论　成功克隆恶性疟原虫FCC1/HN株FEN 1基因。序列测定及同源性分析表明,恶性疟原虫FCC1/HN株与国外已报道各株的FEN 1基因序列有高度同源性。

CLONING AND SEQUENCE ANALYZING OF FEN-1 GENE OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN

Objective To clone and determine the nucleotide sequences of flap endonuclease (FEN 1), and analyze the differences of the sequences of FEN 1 genes between Plasmodium falciparum isolate FCC1/HN and other ones worldwide. Methods The FEN 1 gene was amplified by PCR technique from genomic DNA of isolate FCC1/HN. Then it was cloned into PMD18 T vector. The positive clones were screened and identified by agarose gel electrophoresis and endonuclease digestion. And the DNAMAN software were used to find the differences of the sequences of the FEN 1 genes among different P. falciparum isolates. Results The FEN 1 gene of isolate FCC1/HN was specifically amplified, and the correct recombinant plasmid PMD18 T FEN 1 was constructed. The result of sequencing the nucleotides showed that the FEN 1 gene of isolate FCC1/HN was 1 947 bp in full length without intron, coding 648 amino acids. Compared with Gambia isolate, the FEN 1 gene of FCC1/HN isolate has 6 base pairs deletion at 1 581- 1 586 bp and other 5 bp mutations. Conclusion The FEN 1 gene of P. falciparum isolate FCC1/HN was successfully cloned and sequenced. Sequences analysis showed that the deduced amino acids of the FEN 1 genes of isolates FCC1/HN and other one shared quite high homology.