恶性疟原虫海南株(FCC1/HN)乳酸脱氢酶基因的分子克隆及序列分析

Cloning and sequencing analysis of the gene coding for the lactate dehydrogenase of Plasmodium falciparum FCC1/HN isolate

目的 了解恶性疟原虫海南株 (FCC1/HN)乳酸脱氢酶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)LDH基因全编码区 ,产物经EcoRⅠ +SalⅠ酶切后 ,定向克隆至pGEX - 4T - 1表达质粒 ,采用Sanger双脱氧链末端终止法测序 ,与其它生物LDH进行同源性分析。 结果 成功地扩增了恶性疟原虫海南株 (FCC1/HN)LDH编码基因 ,大小为 95 1bp ,无内含子 ,与Honduras株LDH基因相比 ,两者有 5个碱基不同 ,推导的氨基酸序列有 4个氨基酸残基不同 ,与刚地弓形虫、隐孢子虫和芽孢杆菌的同源性分别为 49.0 6 % (15 6 / 318)、42 .5 8% (132 / 310 )、31.6 1% (98/310 )。与人的LDH -A、LDH -B和LDH -C的同源性分别为 30 .19% (93/ 30 8)、2 9.5 5 % (91/ 30 8)和 33.44 % (10 4/ 311)。结论 FCC1/HN株与Honduras株LDH高度同源 。

Aim To analyze the complete sequences of lactate dehydrogenase(LDH) gene from Plasmodium falciparum FCC1/HN isolate.Methods The complete gene coding for LDH of Plasmodium falciparum FCC1/HN isolate was amplified by PCR.PCR products were digested by EcoRⅠ/SaIⅠ and cloned into plasmid PGEX 4T 1.Recombinant plasmids PGEX LDH were screened and identified by PCR and restriction analysis.The cloned LDH gene was then sequenced by the use of Sanger's method.Homology of LDHs among Plasmodium falciparum FCC1/HN isolate,Honduras isolate and other species were analysed.Result LDH gene of Plasmodium falciparum FCC1/HN isolate was successfully amplified and cloned into the PGEX 4T 1 vector.DNA sequencing analysis showed that the coding length of gene was 951bp,without any introns.By comparing to the LDH gene of Honduras isolate it was found that there are five point mutations ccurred in the LDH gene of FCC1/HN isolate and four point mutations based of deduced amino acid sequences.LDH of FCC1/HN isolate exhibited 49.06%,42.58%,31.61% homologies in amino acids with Toxoplasma gondii,Cryptosporidium parvum,Bacillus megaterium,and 30.19%,29.55%,33.44% homology in amino acids with human LDH A,LDH B,LDH C respectively.Conclusion The coding region of LDH gene of Plasmodium falciparum FCC1/HN isolate was highly homologous with Honduras isolate.It is very clear that Plasmodium LDH gene is very different from the LDH isozymes of other origins.