间日疟原虫裂殖子表面蛋白1和环子孢子蛋白基因多态性分析

doi:10.12140/j.issn.1000-7423.2020.03.011

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (3): 323-331.doi: 10.12140/j.issn.1000-7423.2020.03.011

间日疟原虫裂殖子表面蛋白1和环子孢子蛋白基因多态性分析

金行一¹, 张玲玲², 朱素娟¹, 徐卫民¹, 陈珺芳¹, 阮卫³, 姚立农³, 陈华良³^,^*()

1 杭州市疾病预防控制中心,杭州 310021
2 南京市鼓楼区疾病预防控制中心,南京 210003
3 浙江省疾病预防控制中心,杭州 310051

收稿日期:2019-11-20 出版日期:2020-06-30 发布日期:2020-07-07
通讯作者: 陈华良
作者简介:金行一(1965-),女,大专,主任技师,从事寄生虫病检验与防治研究。E-mail: 1499256186@qq.com
基金资助:
杭州市农业与社会发展科研主动设计项目(20190101A12);杭州市卫生计生科技计划项目(2018A79)

Analysis of genetic polymorphisms of merozoite surface protein-1 and circumsporozoite protein of Plasmodium vivax

JIN Hang-yi¹, ZHANG Ling-ling², ZHU Su-juan¹, XU Wei-min¹, CHEN Jun-fang¹, RUAN Wei³, YAO Li-nong³, CHEN Hua-liang³^,^*()

1 Hangzhou Center for Disease Control and Prevention, Hangzhou 310021, China
2 Nanjing Gulou County Center for Disease Control and Prevention, Nanjing 210003, China
3 Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China

Received:2019-11-20 Online:2020-06-30 Published:2020-07-07
Contact: Hua-liang CHEN
Supported by:
Hangzhou Agricultural and Social Development Research Initiative Design Project(20190101A12);Hangzhou Health and Family Planning Science and Technology Project(2018A79)

摘要/Abstract

摘要：

目的对杭州市间日疟病例感染的间日疟原虫（Plasmodium vivax）裂殖子表面蛋白1（MSP-1）和环子孢子蛋白（CSP）基因的多态性进行描述性分析。方法收集杭州市2008-2019年报告的间日疟病例的血样和流行病学资料。采用全血DNA抽提试剂盒提取疟原虫基因组DNA,巢式PCR分别扩增Pvmsp-1和Pvcsp基因的特异性片段,并进行双向测序。应用DNAStar、MEGA 6.0软件对氨基酸序列进行序列比对和遗传进化分析,并采用邻接法构建系统进化树。采用SPSS 17.0统计学软件对基因多态性及感染来源地进行描述性和归纳分析。结果共收集间日疟病例血样56份,巢式PCR扩增的Pvmsp-1和Pvcsp基因片段分别测序成功44份和54份血样。序列分析显示,44条Pvmsp-1序列可分为4种基因型,分别为SalⅠ型18份、Belem型8份、R-Ⅲ型17份和R-Ⅳ型1份;54条Pvcsp序列分为两种基因型,分别为VK210型52份和VK247型2份。对Pvmsp-1基因的分型特征和感染来源地分析发现,SalⅠ型分布广泛,在我国华东地区,东南亚地区,印度,埃塞俄比亚等多个国家或地区存在。我国华东地区的主要分布类型为R-Ⅲ型,此外,R-Ⅲ型在南亚、东南亚地区也有分布。Belem型主要分布在东南亚、南亚和埃塞俄比亚等国家或地区。对Pvcsp基因的多态性特征和感染来源地分析发现,共有4种多态性特征具有一定的地域特征。结论杭州市2008-2019年报告的间日疟病例的Pvmsp-1和Pvcsp基因存在较丰富的多态性特征,部分特征具有一定地域性。

关键词: 间日疟原虫, 裂殖子表面蛋白1, 环子孢子蛋白, 基因多态性

Abstract:

Objective This study aimed to conduct descriptive analysis of the genetic polymorphisms of Plasmodium vivax gene merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) in vivax malaria cases in Hangzhou City. Methods Blood samples and epidemiological information of vivax malaria cases reported in Hangzhou during 2008-2019 were collected. P. vivax genomic DNA was extracted by QIAamp DNA blood mini kit. Pvmsp-1 and Pvcsp were amplified by nested PCR and the amplification products were sequenced. The amino-acid sequences were analyzed using DNAStar and MEGA 6.0 softwares and the phylogenetic tree was constructed using the neighbor-joining method. The descriptive and inductive analysis of genetic polymorphism and place of infection origin were performed using SPSS 17.0. Results Fifty-six vivax malaria cases were sampled, of whom 44 and 54 samples were successfully sequenced for Pvmsp-1 and Pvcsp, respectively. Sequence analysis indicated that 44 Pvmsp-1 sequences were clustered into four genotypes: SalⅠ(18 samples), Belem (8), R-Ⅲ (17) and R-Ⅳ (1); 54 Pvcsp sequences were devided into two genotypes: VK210 (52 samples) and VK247 (2). Analysis of the genotyping and place of infection origin of Pvmsp-1 gene showed that the SalⅠtype was widely distributed in East China, Southeast Asia, India, and Ethiopia. The main type of Pvmsp-1 in East China was R-Ⅲ. In addition, the R-Ⅲ type was also distributed in South Asia and Southeast Asia. The Belem type was mainly distributed in Southeast Asia, South Asia and Ethiopia. Analysis of Pvcsp gene polymorphism and place of infection origin revealed four types of polymorphism with some regional features. Conclusion The Pvmsp-1 and Pvcsp genes in vivax malaria cases reported in Hangzhou during 2008-2019 were characterized being highly polymorphic, with some genotypes having certain regional features.

Key words: Plasmodium vivax, Merozoite surface protein-1, Circumsporozoite protein, Genetic diversity

中图分类号:

R531.31

金行一, 张玲玲, 朱素娟, 徐卫民, 陈珺芳, 阮卫, 姚立农, 陈华良. 间日疟原虫裂殖子表面蛋白1和环子孢子蛋白基因多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 323-331.

JIN Hang-yi, ZHANG Ling-ling, ZHU Su-juan, XU Wei-min, CHEN Jun-fang, RUAN Wei, YAO Li-nong, CHEN Hua-liang. Analysis of genetic polymorphisms of merozoite surface protein-1 and circumsporozoite protein of Plasmodium vivax[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(3): 323-331.

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基因 Genes	引物名称 Primer names	引物序列（5′→3′） Primer sequences (5′→3′)
Pvmsp-1	Pvmsp-1-F1	CCCTACTACTTGATGGTCCTC
	Pvmsp-1-R1	CCTTCTGGTACAGCTCAATG
	Pvmsp-1-F2	AGCATGATCGCCACTGAGAAG
	Pvmsp-1-R2	GTGCTTGTGACATGCGTAAGC
Pvcsp	VCSF1	ATGTAGATCTGTCCAAGGCCATAAA
	VCSR1	TAATTGAATAATGCTAGGACTAACAATATG
	VCSF2	GCAGAACCAAAAAATCCACGTGAAAATAAG
	VCSR2	CCAACGGTAGCTCTAACTTTATCTAGGTAT

来源地 Source area	Pvmsp-1/Pvcsp												合计Total
来源地 Source area	2008	2009	2010	2011	2012	2013	2014	2015	2016	2017	2018	2019	合计Total
中国安徽Anhui, China	1, 2	0, 0	4, 4	4, 4	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	9, 10
中国浙江Zhejiang, China	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
中国四川Sichuan, China	0, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 1
柬埔寨Cambodia	0, 0	0, 0	0, 0	2, 2	2, 3	2, 2	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	6, 7
缅甸Myanmar	0, 0	0, 0	0, 0	0, 1	0, 1	0, 0	0, 0	2, 2	0, 0	0, 0	0, 0	0, 0	2, 4
印度尼西亚Indonesia	0, 0	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
印度India	0, 0	0, 0	0, 0	2, 2	0, 0	0, 0	1, 1	0, 0	0, 0	1, 1	0, 0	0, 0	4, 4
巴基斯坦Pakistan	0, 0	0, 0	0, 0	2, 3	0, 0	1, 2	0, 0	1, 1	1, 3	1, 1	0, 0	1, 1	7, 11
阿富汗Afghanistan	0, 0	0, 0	0, 0	0, 0	0, 0	2, 2	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	2, 2
埃塞俄比亚Ethiopia	0, 0	0, 0	0, 0	0, 1	0, 0	0, 0	0, 0	2, 2	0, 0	3, 3	0, 0	0, 0	5, 6
尼日利亚Nigeria	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1	2, 2
苏丹Sudan	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
科特迪瓦Cote d′ivoire	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 1	0, 0	0, 1
非洲（不详）Africa	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
未知来源地Unknown	0, 0	0, 0	2, 1	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	3, 2
合计Total	1, 3	2, 2	6, 5	12, 15	3, 5	6, 7	1, 1	5, 5	1, 3	5, 5	0, 1	2, 2	44, 54

来源地Area	V1				V2		V3				V4						V5
来源地Area	a	b	c	d	e	f	g	h	i	j	k	l	m	n	o	p	q	r	s	t	u	v	w
中国安徽Anhui, China	1	0	8	0	10	0	0	10	0	0	0	1	0	3	4	2	0	2	0	1	3	4	0
中国浙江Zhejiang, China	0	0	1	0	1	0	0	1	0	0	0	1	0	0	0	0	0	0	0	0	0	0	1
中国四川Sichuan, China	0	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
柬埔寨Cambodia	3	1	2	0	5	2	4	1	1	1	0	2	4	1	0	0	1	5	1	0	0	0	0
缅甸Myanmar	2	0	0	0	1	1	0	1	1	0	0	1	0	1	0	0	0	1	1	0	0	0	0
印度尼西亚Indonesia	0	1	0	0	0	1	0	0	1	0	0	0	0	1	0	0	0	1	0	0	0	0	0
印度India	4	0	0	0	4	0	4	0	0	0	1	3	0	0	0	0	0	4	0	0	0	0	0
巴基斯坦Pakistan	3	1	3	0	11	0	11	0	0	0	0	11	0	0	0	0	0	11	0	0	0	0	0
阿富汗Afghanistan	0	0	2	0	2	0	2	0	0	0	0	2	0	0	0	0	0	2	0	0	0	0	0
埃塞俄比亚Ethiopia	1	3	1	0	6	0	6	0	0	0	0	6	0	0	0	0	0	6	0	0	0	0	0
尼日利亚Nigeria	1	1	0	0	2	0	2	0	0	0	0	2	0	0	0	0	0	2	0	0	0	0	0
苏丹Sudan	1	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
科特迪瓦Cote d’ivoire	0	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
非洲（不详）Africa	0	0	0	1	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
未知Unknown	2	1	0	0	2	0	1	1	0	0	0	1	0	0	0	1	0	1	0	0	1	0	0

基因 Gene	多态性特征 Polymorphism characteristics	地域分布 Regional distribution
基因 Gene	多态性特征 Polymorphism characteristics	中国华东地区 East China	东南亚地区 Southeast Asia	南亚地区 South Asia	非洲地区 Africa
Pvmsp-1	SalⅠ	√	√	√	√
	Belem		√	√	√
	R-Ⅲ	√	√	√	√
	R-Ⅳ				√
Pvcsp	中央重复区后不具有12肽插入序列 Without a 12-peptide insertion sequence after the central repeat region		√
	12肽插入序列后GGNA重复次数 Number of repeats of the GGNA unit after the 12-peptide insertion sequence	2	1~4	1	1
	中央重复区9肽单位重复次数 Number of repeats of peptide repeat motif in central repeat region	18~22	18~20	18	18
	9肽单位（GNGAGGQAA）重复次数 Number of repeats of peptide repeat motif (GNGAGGQAA)	1, 13~15	1~2	1	1

间日疟原虫裂殖子表面蛋白1和环子孢子蛋白基因多态性分析

Analysis of genetic polymorphisms of merozoite surface protein-1 and circumsporozoite protein of Plasmodium vivax

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