河南省输入性卵形疟原虫2个亚种富色氨酸抗原基因的多态性分析

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (1): 13-17.

河南省输入性卵形疟原虫2个亚种富色氨酸抗原基因的多态性分析

周瑞敏^*(), 李素华, 张雅兰, 钱丹, 杨成运, 刘颖, 赵玉玲, 张红卫, 许汴利

河南省疾病预防控制中心,郑州 450016

收稿日期:2016-07-11 出版日期:2017-02-28 发布日期:2017-03-06
通讯作者: 周瑞敏
基金资助:
河南省医学科技攻关计划项目（No. 201304053,201602318）

Polymorphisms of tryptophan-rich antigen gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province

Rui-min ZHOU^*(), Su-hua LI, Ya-lan ZHANG, Dan QIAN, Cheng-yun YANG, Ying LIU, Yu-ling ZHAO, Hong-wei ZHANG, Bian-li XU

Henan Centre for Disease Control and Prevention, Zhengzhou 450016, China

Received:2016-07-11 Online:2017-02-28 Published:2017-03-06
Contact: Rui-min ZHOU

摘要/Abstract

摘要：

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,PoTRA）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PoTRA基因,连接至pMD18-T载体,提取质粒进行测序,在GenBank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PoTRA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%,PoTRA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%,PoTRA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PoTRA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。

关键词: 输入性卵形疟, 卵形疟原虫curtisi亚种, 卵形疟原虫wallikeri亚种, 富色氨酸抗原

Abstract:

Objective To analyze the genetic sequence of tryptophan-rich antigen (PoTRA) gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province in 2015. Methods Blood samples were collected from 22 imported ovale malaria cases in Henan Province in 2015. After DNA extraction, PoTRA was amplified by nested PCR, and was inserted into the pMD18-T vector. The plasmid was extracted and sequenced, and the results were blasted in GenBank to determine the subspecies of P. ovale. The sizes and species of the PoTRA gene were analyzed. The amino-acid sequence of PoTRA was also aligned to analyze the difference in amino acid sequence. The phylogenetic tree was constructed to analyze the genetic relationship among the samples by neighbor-joining. Results Of the 22 imported cases, eight (36.4%) were infected with P. ovale wallikeri, which had two sizes, the predominant 245 and 299 bp. The other 14 cases (63.6%) were infected with P. ovale curtisi, which had three sizes, the predominant 299, 317 and 335 bp. Amino-acid sequence alignment revealed that the two types of P. ovale wallikeri differed in two amino-acid units, MANPINMANPIN and AITPIN, while the three types of P. ovale curtisi differed in amino-acid units TITPIS and TINPIN. The phylogenetic tree showed that the 22 samples belonged to two subpopulations of P. ovale curtisi and P. ovale wallikeri, wherein the P. ovale curtisi was further divided into two sub-branches, and samples with sizes of 317 and 335 bp were in the same sub-branch with a closer genetic relationship. Conclusion Two subspecies, P. ovale curtisi and P. ovale wallikeri, are identified from the imported ovale malaria cases in Henan Province in 2015. The P. ovale curtisi has three genetic types and P. ovale wallikeri has two genetic types of PoTRA gene, revealing genetic polymorphisms of PoTRA.

Key words: Imported ovale malaria, P. ovale curtisi, P. ovale wallikeri, Tryptophan-rich antigen

中图分类号:

R382.319

周瑞敏, 李素华, 张雅兰, 钱丹, 杨成运, 刘颖, 赵玉玲, 张红卫, 许汴利. 河南省输入性卵形疟原虫2个亚种富色氨酸抗原基因的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 13-17.

Rui-min ZHOU, Su-hua LI, Ya-lan ZHANG, Dan QIAN, Cheng-yun YANG, Ying LIU, Yu-ling ZHAO, Hong-wei ZHANG, Bian-li XU. Polymorphisms of tryptophan-rich antigen gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 13-17.

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参考文献 16

[1]	Stephens JW.A new malaria parasite of man[J]. Ann Trop Med Parasitol, 1922, 16(4):383-388.
[2]	Win TT, Lin K, Mizuno S, et al. Wide distribution of Plasmodium ovale in Myanmar[J]. Trop Med Int Health, 2002, 7(3): 231-239.
[3]	Kawamoto F, Liu Q, Ferreira MU, et al. How prevalent are Plasmodium ovale and P. malariae in East Asia[J]. Parasitol Today (Regul Ed), 1999, 15(10): 422-426.
[4]	Petersen E, Høgh B, Marbiah NT, et al. A longitudinal study of antibodies to the Plasmodium falciparum antigen Pf155/RESA and immunity to malaria infection in adult Liberians[J]. Trans R Soc Trop Med Hyg, 1990, 84(3): 339-345.
[5]	张丽, 周水森, 丰俊, 等. 2014年全国疟疾疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(5): 319-326.
[6]	张丽, 丰俊, 张少森, 等. 2015年全国疟疾疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 477-481.
[7]	丰俊, 夏志贵. 2004-2013年中国疟疾发病情况及趋势分析[J]. 中国病原生物学杂志, 2014, 9(5): 442-446.
[8]	Snounou G, Viriyakosol S, Zhu XP, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction[J]. Mol Biochem Parasitol, 1993, 61(2): 315-320.
[9]	Kawamoto F, Miyake H, Kaneko O, et al. Sequence variation in the 18S rRNA gene, a target for PCR-based malaria diagnosis, in Plasmodium ovale from southern Vietnam[J]. J Clin Microbiol, 1996, 34(9): 2287-2289.
[10]	Sutherland CJ, Tanomsing N, Nolder D, et al. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally[J]. J Infect Dis, 2010, 201(10): 1544-1550.
[11]	Oguike MC, Betson M, Burke M, et al. Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities[J]. Int J Parasitol, 2011, 41(6): 677-683.
[12]	Fuehrer HP, Noedl H.Recent advances in detection of Plasmodium ovale: implications of separation into the two species Plasmodium ovale wallikeri and Plasmodium ovale curtisi[J]. J Clin Microbiol, 2014, 52(2): 387-391.
[13]	Calderaro A, Piccolo G, Perandin F, et al. Genetic polymorphisms influence Plasmodium ovale PCR detection accuracy[J]. J Clin Microbiol, 2007, 45(5): 1624-1627.
[14]	李美, 夏志贵, 汤林华. 卵形疟原虫wallikeri亚种及其基因检测体系的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(1): 64-67.
[15]	Li M, Xia Z, Yan H.New type of SSUrDNA sequence was detected from both Plasmodium ovale curtisi and Plasmodium ovale wallikeri samples[J]. Malar J, 2014, 13: 216.
[16]	Tanomsing N, Imwong M, Sutherland CJ, et al. Genetic marker suitable for identification and genotyping of Plasmodium ovale curtisi and Plasmodium ovale wallikeri[J]. J Clin Microbiol, 2013, 51(12): 4213-4216.

样本编号 Sample code	国家 Country	卵形疟亚种 P. ovale subspecies	PoTRA长度/bp Size of PoTRA/bp
Po01	安哥拉	wallikeri	245
Po02	安哥拉	wallikeri	245
Po03	安哥拉	wallikeri	245
Po04	喀麦隆	wallikeri	245
Po05	喀麦隆	wallikeri	245
Po06	刚果	wallikeri	299
Po07	尼日利亚	wallikeri	299
Po08	尼日利亚	wallikeri	245
Po09	安哥拉	curtisi	299
Po10	尼日利亚	curtisi	299
Po11	尼日利亚	curtisi	299
Po12	赤道几内亚	curtisi	299
Po13	赤道几内亚	curtisi	299
Po14	加纳	curtisi	299
Po15	加纳	curtisi	299
Po16	利比里亚	curtisi	299
Po17	坦桑尼亚	curtisi	299
Po18	安哥拉	curtisi	317
Po19	尼日利亚	curtisi	317
Po20	刚果	curtisi	317
Po21	几内亚	curtisi	317
Po22	喀麦隆	curtisi	335