摘要
目的分析云南省不同感染来源恶性疟原虫裂殖子表面蛋白3(PfMSP-3)基因和抗原表位多态性。方法收集2012年8月-2016年12月寄生虫病防治信息管理系统登记报告的云南省本地和输入恶性疟病例的血样和流行病学史等信息。提取血样的疟原虫DNA,半巢式PCR扩增PfMSP-3基因并测序,与Gen Bank中的恶性疟原虫Pf3D7、PfK1分离株的参比序列LN999944.1、U08851.1进行比对。用MEGA 5.04、Arlequin 3.5.2.2分析PfMSP-3基因的单倍型、期望杂合度(He)、群体间遗传分化指数(Fst)。用Network 4.6.0构建单倍型网络进化图。用Dna SP 5.10计算核苷酸的非同义(Ka)、同义置换率(Ks)。用IEDB在线预测软件预测PfMSP-3肽链T细胞、B细胞抗原表位。结果共收集190份恶性疟病例血样,其中181份扩增出长1 100 bp的PfMSP-3片段,测序成功167份(缅甸121份、非洲37份、云南本地感染9份)。序列比对结果显示,167条DNA序列存在36种单倍型,He为0.409±0.183,Ka/Ks为0.98。各单倍型沿Pf3D7型(Ⅰ)、过渡型(Ⅱ)及PfK1型(Ⅲ)等3种方向进化:H_1、H_9等7种单倍型为Pf3D7型,H_7、H_8等6种单倍型为过渡型,H_2、H_3等23种单倍型为PfK1型。Pf3D7型、过渡型和PfK1型序列的占比分别为49.7%(83/167)、12.6%(21/167)和37.7%(63/167)。PfMSP-3基因在非洲与缅甸恶性疟原虫群体间的分化程度最高,Fst=0.049;在非洲与云南本地感染恶性疟原虫群体间最小,Fst=0.032。抗原表位分析结果显示,36种单倍型的PfMSP-3肽链亲水性高于疏水性,指数分别为1.050~2.535和-2.583^-3.544。T细胞抗原表位活性为-1.1;B细胞抗原表位活性平均>0.5,且表现为Pf3D7型>过渡型>PfK1型。结论云南省不同感染来源恶性疟原虫的PfMSP-3基因存在3类基因型,不同基因型PfMSP-3蛋白B细胞抗原表位活性的预测值较T细胞高。
Objective To analyze the gene sequence polymorphisms and predict antigen epitope of Plasmodium falciparum merozoite surface protein-3（ PfMSP-3） from different infection sources of P. falciparum in Yunnan Province. Methods Blood samples on filter paper and the corresponding epidemiology data of patients with falciparum malaria reported through the Reporting System of Chinese Center for Disease Control and Prevention, were collected from August 2012 to December 2016. P. falciparum DNA was extracted, and the PfMSP-3 gene was amplified with semi-nest PCR. PCR products were sequenced, and all sequences were aligned with the reference sequences LN999944.1 of Pf3 D7 isolate and U08851.1 of PfK1 isolate in Gen Bank. The haplotype number, expected heterozygsity（He）, and genetic differentiation（Fst） of PfMSP-3 gene in different Plasmodium populations were analyzed by MEGA 5.04 and Arlequin 3.5.2.2 softwares. The haplotype media evolution tree was constructed with the Network 4.6.0 software. The mean number of non-synonymous substitutions per non-synonymous site（Ka） and synonymous mutations per synonymous site（Ks） were calculated with the Dna SP 5.10 software. T/B cell epitopes of PfMSP-3 peptides were analyzed and predicted using the IEDB online software. Results A total of 190 blood samples were collected, of which 181 produced PfMSP-3 fragments at 1 100 bp, and 167 PCR products were sequenced successfully, including 121 from Myanmar, 37 from Africa and 9 from indigenous cases in Yunnan.Alignment results showed that there were 36 haplotypes in the 167 DNA sequences, with an He of（0.409 ＋ 0.183）and a Ka/Ks of 0.98. The 36 haplotypes evolved along the direction from Pf3 D7 type（ Ⅰ group）, to transitional type（ Ⅱ group）, then PfK1（ Ⅲ group）. Among them, 7 haplotypes including H1 and H9 were Pf3 D7 type, 6 haplotypes including H7 and H8 were the transitional type, and 23 haplotypes including H2 and H3 were the PfK1 type. The Pf3 D7 type, transitional type and PfK1 type accounted for 49.7%（83/167）, 12.6%（21/167） and37.7%（63/167）, respectively. The genetic differentiation of PfMSP-3 was highest between African and Myanmar P.falciparum populations（Fst = 0.049） and lowest between African and Yunnan populations（Fst = 0.032）. The peptide chains of 36 haplotypes of PfMSP-3 had a high hydrophilicity than hydrophobicity, with indexes of 1.050-2.535 and-2.583 -3.544, respectively. The activity score of T cell epitope was-1.1. The average activity score of B cells was 0.5,more specifically ranked as Pf3 D7 type transitional type PfK1 type. Conclusion The PfMSP-3 gene of P.falciparum populations from different infection sources has evolved into 3 genotypes, and the B cell epitopes are predicted to be more active than T cell epitopes in PfMSP-3 proteins of all genotypes.
作者
董莹
邓艳
徐艳春
陈梦妮
毛祥华
孙艾明
王剑
DONG Ying;DENG Yahl;XU Yan-chun;CHEN Meng-ni;MAO Xiang-hua;SUN Ai-ming;WANG Jian(Yunnan Institute of Parasitic Diseases, Yunnan Center of Malaria Research, Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research, Puer 665000, China;Hubei International Travel Healthcare Center, Wuhan 430000, China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2018年第3期210-217,共8页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.81220108019
No.81660559)
云南省科技项目应用基础研究计划青年项目(No.2017FD007
No.2017FD190)~~
关键词
恶性疟原虫
裂殖子表面蛋白3
抗原表位
多态性
云南省
Plasmodium falciparum
Merozoite surface protein-3
Antigen epitope
Polymorphism
Yunnan Province
作者简介
通讯作者:董莹,E-mail:luxidongying@126.com
