云南恶性疟病例疟原虫Pfhrp2基因序列多态性分析董莹1|孙艾明1|2|陈梦妮1|徐艳春1|毛祥华1|邓艳1|杨恒林1*

目的 目的 分析云南省恶性疟病例疟原虫虫株的富组氨酸蛋白Ⅱ （Histidine?rich protein?2，HRPⅡ） 基因 （Pfhrp2） 序列的多态性， 为研究疟原虫抗原基因缺失奠定基础。方法 方法 搜集2012年8月-2015年9月云南省恶性疟现症病例的滤纸血样和相关信息， 用PCR技术扩增血样中恶性疟原虫Pfhrp2基因exon2区并测序， 测序成功序列与AY816237、AY816240、 AY816301参比序列比对； 用Mega 5.04分析Pfhrp2基因exon2区序列多态性， 计算序列间保守位点、 遗传距离等； 根据氨基酸序列间遗传距离绘制聚类树状图。结果 结果 共收集云南省15个州 （市） 的恶性疟现症病例血样218份， 病例感染来源地包括云南本地、 非洲、 缅甸等流行区。其中155份Pfhrp2基因exon2区扩增阳性和测序成功， 编码氨基酸数在115～298之间， 平均为239.7 aa， 非洲 （239.9 aa）、 缅甸 （239.5 aa）、 云南 （241.6 aa） 感染虫株的氨基酸残基均数差异无统计学意义 （F = 0.025， P > 0.05）。所有氨基酸残基序列均以12型重复作为结尾， 以1型、 2型重复为起始， 比例各占98.1%（152/155） 和1.9% （3/155）， 2型重复次数最多， 为12.9次。155条Pfhrp2 基因exon2区DNA序列的同源位点为894 bp， 保守位点占20.8% （186/894）， 变异位点占78.2% （699/894）， 非洲虫株序列间遗传距离为0.000～0.741， 缅甸为0.000～0.948， 云南为0.000～0.750。所有155条序列按氨基酸序列大小聚类成3个大类， 同一个层级的序列具有近似的序列长度和氨基酸重复类型。结论 结论 云南省恶性疟现症病例所感染疟原虫的Pfhrp2基因exon2区存在高度多态性， 恶性疟原虫株主要按Pfhrp2基因exon2区的氨基酸序列长度进行聚类。

Analysis of polymorphism of Pfhrp2 gene in Plasmodium falciparum from falciparum malaria patients in Yunnan Province

Objective Objective To analyze the polymorphism of histidine rich protein 2（HRP II）gene in Plasmodium falciparum（Pfhrp2）from falciparum malaria patients in Yunnan Province，so as to lay the foundation for studying the defection of antigengenes of Plasmodium. Methods Methods The filter paper blood samples and related information of falciparum malaria cases reportedwere obtained in Yunnan Province from August 2012 to September 2015. Under the guidance of the specific primers，the exon2regions in Pfhrp2 gene in P. falciparum from DNA samples were amplified by PCR，and the PCR products were sequenced. Thesequences of exon2 region in Pfhrp2 gene were blasted by comparing with the reference sequences AY816237，AY816240，andAY816301. Next，the polymorphism of the sequence in exon2 region of Pfhrp2 gene was analyzed by MEGA 5.04 software. Theconserved sites and genetic distances between sequences were calculated by using the software as well，and the clustering treewas drawn according to the genetic distances between the amino acid sequences. Results Results A total of 218 bloods samples fromthe falciparum malaria cases in 15 prefectures of Yunnan Province were collected，and the sources of infection included Yun?nan，Africa and Myanmar. The PCR results showed that the exon2 regions in Pfhrp2 genes of 155 samples were positive by am?plification and their products were sequenced successfully. The sequence analysis showed that the length range of the amino acidresidues of exon2 region in Pfhrp2 gene was from 115 aa to 298 aa，the average length was 239.7 aa. There was no statisticallysignificance among the means of the amino acid residues of the isolates from Africa（239.9 aa），Myanmar （239.5 aa）and Yun?nan（241.6 aa） （F = 0.025，P > 0.05） . All the 155 amino acid sequences ended with type 12 repeat，98.1%（152/155）of them started with type 1 repeat and 1.9%（3/155）of them started with type 2. Type 2 presented most frequently repeat in all the se?quences and the average repeat times were 12.9. The homologous locus of the DNA sequences in exon2 regions of the 155 Pfhrp2genes was 894 bp，among which the conservative sites accounted for 20.6%（186/894），and the variable sites for 78.2%（699/894） . The genetic distances between the sequences of Africa isolates ranged from 0 to 0.741，and those of the Myanmar and Yun?nan isolates were 0-0.948 and 0-0.750，respectively. The cluster analysis showed that all the 155 sequences clustered into 3 cat?egories on genetic distances between amino acid sequences according to the size of the amino acid sequence length. At the samelevel，the sequences had approximate lengths and amino acid repeat types. Conclusion Conclusion The sequence of exon2 region inPfhrp2 gene of P. falciparum from falciparum malaria cases in Yunnan Province is highly polymorphic，the P. falciparum iso?lates are clustered mainly according to the size of the amino acid sequence of exon2 region in Pfhrp2 gene.