恶性疟原虫海南株(FCC1/HN)谷氨酸脱氢酶基因的克隆及序列分析

Cloning and sequence analysis of a gene encoding glutamate dehydrogenase of Plasmodium falciparum FCC1/HN isolate

目的 了解恶性疟原虫海南株（ＦＣＣ１／ＨＮ）谷氨酸脱氢酶（ＧＤＨ）基因全编码区核苷酸序列变异情况。方法ＰＣＲ扩增恶性疟原虫海南株（ＦＣＣ１／ＨＮ）ＣＤＨ基因全编码区，产物经ＥｃｏＲＩ＋ＳａｌＩ酶切后，定向克隆至ｐＧＥＸ－４Ｔ－１质粒，采用Ｓａｎｇｅｒ双脱氧链末端终止法测序，与其它生物ＧＤＨ进行同源性分析。结果 成功扩增了恶性疟原虫海南株（ＦＣＣ１／ＨＮ）ＧＤＨ编码基因，大小为１４１０ｂｐ，无内含子，与Ｔｈａｉｌａｎｄ株ＧＤＨ基因相比，两者有２个碱基不同，推导的氨基酸序列完全相同；与蓝氏贾第鞭毛虫、克鲁氏锥虫和梭状芽孢杆菌ＧＤＨ的同源性分别为５７．９０％（２６０／４４９）、５６．５１％（２４３／４３０）、４２．０５％（１６４／３９０）；与人的ＣＤＨ－１、ＧＤＨ－２的同源性分别为２９．０２％（１０１／３４８）、２８．７３％（１０／３４８）。结论 ＦＣＣ１／ＨＮ株与Ｔｈａｉｌａｎｄ株ＧＤＨ高度同源，疟原虫ＧＤＨ明显不同于其它生物ＧＤＨ．

Aim To analyze the complete sequences of glutamate dehydrogenase(GDH) gene from Plasmodium falciparum FCC 1 /HN isolate. Methods The complete gene encoding GDH of Plasmodium falciparum FCC1 /HN isolate was amplified by PCR. PCR products were digested by EcoRI/ SalI and cloned into plasmid pGEX-4T-1. The recombinant plasmid pGEX- GDH was screened and identified by PCR and restriction analysis. The cloned GDH genes were sequenced by Sanger' s method. Homology of GDHs among Plasmodium falciparum FCC1 /HN isolate, Thailand isolate and other species were analysed. Result GDH genes of Plasmodium falciparum FCC 1 / HN isolate were successfully amplified and cloned into plasmid pGEX -4T-1. DNA sequencing showed that the size of gene was 1410bp without introns. By comparing with Thailand isolate GDH gene, there were only two point mutations. The two deduced amino acid sequences were completely the same. GDH of FCC1 /HN isolate exhibited 57. 90%, 56. 51%, 42. 05% homology in amino acids with Giardia lamblia, Trypanosoma cruzi, Clostridium symbiosum, and 29. 02%, 28. 73% homology in amino acids with human GDH- 1, GDH-2, respectively. Conclusion The encoding region of GDH gene of Plasmodium falciparum FCC1 /HN isolate was highly homologous with Thailand isolate. It is obvious that the Plasmodium GDH is most different from the other GDHs.