云南省输入性间日疟原虫多药抗性蛋白1基因突变多态性分析  

Polymorphism analysis of multidrug resistance protein 1 gene in imported Plasmodium vivax in Yunnan Province

作　　者：丁红芸 董莹[2] 徐艳春[2] 邓艳[2] 刘言 吴静 陈梦妮[2] 张苍林[2] DING Hongyun;DONG Ying;XU Yanchun;DENG Yan;LIU Yan;WU Jing;CHEN Mengni;ZHANG Canglin(Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,Yunnan,China;Yunnan Institute of Parasitic Diseases,Yunnan Center of Malaria Research,Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research,Puer 665000,China)

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500 [2]云南省寄生虫病防治所,云南省疟疾研究中心,云南省虫媒传染病防控研究重点实验室,普洱665000

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第4期404-411,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(81660559,82160637)。

摘　　要：目的分析云南省输入性间日疟原虫的多药抗性蛋白1基因(Pvmdr1)突变多态性。方法对云南省2020年和2021年诊断为间日疟原虫感染的病例血样进行Pvmdr1全基因扩增及测序,以间日疟原虫Sal‑Ⅰ分离株的序列(GenBank登录号:NC_009915.1)为模板设计PCR反应引物,用MEGA 5.04软件与编码区参比序列(GenBank登录号:XM_001613678.1)进行比对,用DnaSP 6.11.01软件分析单倍型和单核苷酸多态(SNP)位点及其突变类型,并计算核酸多样性指数π、单倍型期望杂合度(He)等,用Network 10.0软件构建中介网络进化图。结果共收集276份输入性间日疟患者血样,其中自缅甸感染病例占99.3%(274/276),自非洲、巴基斯坦感染各占0.4%(1/276)。259份血样扩增获得4392 bp的Pvmdr1全基因序列,提交GenBank获得的登录号为OP559204~OP559462。259条DNA序列π、Ka/Ks比值分别为0.00076和3.6006,共检出22个SNP,包括13个非同义突变和9个同义突变。仅c.4074C>T位点突变在2020、2021年的检出率[15.0%(21/140)、5.9%(7/119)]差异有统计学意义(χ^(2)=5.546,P<0.05)。次等位位点为c.1587A>G(97.9%,253/259),新检出SNP包括c.2499G>T(2.3%,6/259)、c.3358C>T(0.4%,1/259)、c.3832C>T(0.4%,1/259)。c.3064C>T、c.4065A>G、c.3358C>T和c.3832C>T突变位点仅在2021年的患者血样中检出,其中前2个SNP从非洲感染的分离株中检出,后2个SNP从缅甸感染的分离株中检出。259条Pvmdr1完整编码区与参比序列(单倍型Hap_1)比对,识别出26种单倍型(Hap_2~Hap_27),均为参比序列(单倍型Hap_1)的突变型,He为0.8907。其中,Hap_8的检出率最高(18.5%,48/259),非洲感染分离株中仅检出Hap_19。2020、2021年的患者血样中检出的单倍型种类分别有15和22种,当年独有的单倍型分别为4和10种。Hap_10在2020、2021年患者血样中的检出率分别为15.0%(21/140)和3.4%(4/119),差异有统计学意义(χ2=22.264,P<0.05)。不同单倍型的多重突变识别结果显示,4重、5重、6重、7重、9重、10�Objective To analyze the polymorphism of Plasmodium vivax multidrug resistance protein 1 gene(Pvmdr1)from imported vivax malaria patients in Yunnan Province.Methods The whole Pvmdr1 gene was amplified and sequenced in the blood samples of patients who diagnosed with vivax malaria in 2020 and 2021 in Yunnan Province.The sequence of P.vivax SalⅠisolate(GenBank accession number:NC_009915.1)was taken as the reference sequence for designing primers.MEGA 5.04 software was used to align the spliced sequences with the coding region reference sequence(GenBank accession number:XM_001613678.1).Haplotype and single nucleotide polymorphism(SNP)sites and their mutation types,nucleotide diversityπ,expected heterozygosity(He),etc were analyzed by DnaSP 6.11.01.Haplotype medium network diagram was constructed using Network 10.0 Software.Results A total of 276 blood samples were collected from vivax malaria patients and were imported from overseas.The full gene sequences with 4392 bp of Pvmdr1 were obtained from 259 blood samples,and the accession numbers were OP559204-OP559462 assigned by GenBank.Theπof 259 DNA coding sequences was 0.00076.A total of 22 SNPs were detected from 259 coding sequences,including 13 non‑synonymous mutant and 9 synonymous mutant loci.Only the detection rate difference of c.4074C>T mutation between 2020 and 2021[15.0%(21/140),5.9%(7/119)]was statistically significant(χ^(2)=5.546,P<0.05).The minor allele frequency was c.1587A>G(97.9%,253/259),and the newly discovered SNPs included c.2499G>T(2.3%,6/259),c.3358C>T(0.4%,1/259),and c.3832C>T(0.4%,1/259).All of c.3064C>T,c.4065A>G,c.3358C>T and c.3832C>T mutant sites were detected from the blood samples in 2021.The 259 complete coding region sequences of Pvmdr1 were defined as 26 haplotypes(Hap_2-Hap_27)by comparison with the reference sequence(haplotype Hap_1,all of which were mutant type haplotypes,and He was 0.8907.Among them,the detection rate of Hap_8 was the highest(18.5%,48/259).There were 15 and 22 haplotypes found from blood samples in 2020 and

关 键 词：间日疟原虫 多药抗性蛋白1 突变 云南省 

分 类 号：R382.311[医药卫生—医学寄生虫学]