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目的分析河南省2015年输入性卵形疟原虫富色氨酸抗原(Plasmodium ovale tryptophan-rich antigen,Po TRA)基因序列。方法采集河南省2015年22例输入性卵形疟患者的血样,收集患者相关信息。提取血样DNA,用巢式PCR方法扩增Po TRA基因,连接至p MD18-T载体,提取质粒进行测序,在Gen Bank中进行同源性比对,判断卵形疟原虫的亚种,并对其基因长度及种类进行分析。对Po TRA基因的氨基酸序列进行比对,分析氨基酸的差异。用邻接法构建系统进化树,分析样本间的亲缘关系。结果 22例卵形疟中,8例为卵形疟原虫wallikeri亚种(P.ovale wallikeri),占36.4%,Po TRA基因长度有245和299 bp 2种类型,以245 bp为主;14例为卵形疟原虫curtisi亚种(P.ovale curtisi),占63.6%,Po TRA基因长度有299、317和335 bp等3种类型,以299 bp为主。氨基酸序列比对显示,wallikeri亚种的2种基因类型相差2个氨基酸单元(MANPINMANPIN和AITPIN),curtisi亚种的3种基因类型间相差2个氨基酸单元(TITPIS和TINPIN)。系统进化树显示,22例样本分为curtisi和wallikeri 2个亚群,其中curtisi亚群又分为2个亚支,299 bp的样本在同一亚支内,317和335 bp的样本在另一亚支内,亲缘关系更近。结论 2015年河南省输入性卵形疟有curtisi和wallikeri 2个亚种,其Po TRA基因存在多态性,curtisi亚种有3种基因类型,wallikeri亚种有2种基因类型。
Objective To analyze the sequence of Enterotoxigenic Plasmodium ovale tryptophan-rich antigen (Po TRA) in Henan Province in 2015. Methods Blood samples of 22 imported patients with oval-shaped malaria were collected from 2015 in Henan Province, and the related information was collected. The blood DNA was extracted, and the Po TRA gene was amplified by nested PCR and ligated into p MD18-T vector. The plasmids were extracted and sequenced. The homology comparison was performed in Gen Bank to determine the subspecies of Plasmodium ovale. Its gene length and type were analyzed. The amino acid sequence of Po TRA gene was compared to analyze the differences of amino acids. The phylogenetic tree was constructed by using the neighbor-joining method to analyze the genetic relationship among the samples. Results Among the 22 cases of oviparous malaria, 8 were P.ovale wallikeri, accounting for 36.4%. The length of Po TRA gene was 245 bp and 299 bp. It was 63.6% for P.ovale curtisi and 299, 317 and 335 bp for Po TRA gene, with 299 bp. Amino acid sequence alignment revealed two amino acid units (MANPINMANPIN and AITPIN) in the wallikeri subspecies, and two amino acid units (TITPIS and TINPIN) in the curtisi subspecies. Phylogenetic tree showed that 22 samples were divided into two subgroups: curtisi and wallikeri, of which curtisi subgroup was divided into two sub-branches, 299 bp in the same sub-branch, 317 and 335 bp in another sub-group Within the branch, kinship closer. Conclusion There are two subspecies curtisi and wallikeri in Henan Province in 2015, which have polymorphism of Po TRA gene, three gene types of curtisi subspecies and two gene of wallikeri subspecies.