恶性疟原虫FCC1/HN株谷氨酸脱氢酶基因的克隆及序列分析

目的克隆恶性疟原虫海南株(FCC1/HN)谷氨酸脱氢酶(GDH)基因并测定其序列,比较FCC1/HN株与国外分离株GDH基因序列的差异.方法根据GDH基因已知序列设计合成一对引物,应用PCR技术从FCC1/HN株基因组DNA中扩增GDH基因,并将其克隆入pMD18-T载体.阳性克隆的重组质粒经酶切及PCR鉴定后,用双脱氧链末端终止法进行基因序列测定.应用DNAstar软件比较不同分离株GDH基因序列的同源性.结果PCR扩增得到特异的FCC1/HN株GDH基因序列.酶切及PCR鉴定获得了正确的pT-GDH重组质粒.测序表明,恶性疟原虫FCC1/HN株GDH基因全长1 329 bp,编码442个氨基酸.序列分析表明,我国恶性疟原虫FCC1/HN株与国外的FCQ27、K1株GDH基因编码的氨基酸序列有少量的差异.结论克隆了恶性疟原虫FCC1/HN株GDH基因;序列测定及同源性分析表明,FCC1/HN株与其它分离株的GDH基因序列有较高的同源性.

CLONING AND SEQUENCE ANALYZING OF GDH GENE OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN

Objective To clone and determine the nucleotide sequence of the glutamate dehydrogenase (GDH) gene of Plasmodium falciparum isolate FCC1/HN, and analyze the differences of the sequences of GDH genes among isolate FCC1/HN and other ones worldwide. Methods The GDH 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 PCR technique. The cloned GDH gene was determined by the dideoxy chain termination method. And the DNAstar software was used to find out the differences of the sequences of the GDH genes among different P. falciparum isolates. Results The GDH gene of isolate FCC1/HN was specifically amplified, and the correct recombinant plasmid pT GDH was constructed. The result of sequencing the nucleotides showed that the GDH gene of isolate FCC1/HN was 1 329 base pairs in full length, encoding 442 amino acids. P. falciparum isolates FCC1/HN, FCQ27 and K1 have a few different amino acids deduced from the GDH genes. Conclusion The GDH gene of P. falciparum isolate FCC1/HN was successfully cloned and sequenced. Sequences analysis showed that the GDH genes of isolate FCC1/HN and other ones shared high homology.