中缅边境及泰国东南地区恶性疟原虫K13基因侧翼微卫星多态性研究

摘要/Abstract

摘要： 目的研究中缅边境拉咱地区和泰国东南Srisaket、Trat和Ranong等3个地区恶性疟原虫（Plasmodium falciparum）K13基因侧翼微卫星的多态性。方法取前期经基因型研究的恶性疟原虫样品42份,其中中缅边境拉咱地区22份（K13基因R539T突变型和野生型各11份）,泰国3个地区20份（K13基因R539T突变型9份和野生型11份）,对上述样品K13基因上游31 900、6 360、150 bp和下游1 700、11 000、31 500 bp共6个位点的微卫星片段进行PCR扩增,PCR产物经毛细管电泳仪检测片段长度,并采用Bio Calculator软件进行数据分析,GenALEx软件计算等位基因频率和期望杂合度（He）。结果中缅边境拉咱地区和泰国3个地区恶性疟原虫虫株K13基因两侧的6个微卫星位点共检测到46个等位基因,在上游6 360 bp位点的等位基因长度分别集中在286和278 bp,等位基因频率为73%和67%。上游31 900 bp位点等位基因长度分别集中在211和205 bp,等位基因频率为55%和44%。上游150 bp及下游1 700 bp位点等位基因长度主要集中在195和209 bp,其他两个位点未出现集中分布的情况。中缅边境拉咱地区虫株的单倍体型为H1至H4型,泰国3个地区虫株除了2株H2单体型外,其余均为H5和H6型。42份样品的6个微卫星位点的平均He分别为0.49 ± 0.23、0.42 ± 0.33和0.72 ± 0.12、0.67 ± 0.11。R539T突变的虫株在毗邻K13基因的微卫星位点处He明显降低,在上游150 bp处分别为0.17和0,在下游1 700 bp处分别为0.3和0。结论中缅边境拉咱地区及泰国3个地区K13基因为R539T突变型的恶性疟原虫虫株均受到青蒿素选择压力,等位基因型在上游6 360和31 900 bp两个位点上具有明显的差异.

关键词: 恶性疟原虫, 微卫星, K13基因

Abstract: Objective To analyze the polymorphisms of microsatellite loci flanking the K13 gene(K13) of Plasmodium falciparum in Lazan of China-Myanmar border and three areas in Thailand. Methods Forty-two P. falciparum samples were collected, including 22 from Lazan(11 with R539T mutation and 11 wild type) and 20 from three areas of Thailand(9 with R539T mutation and 11 wild type). Six microsatellite loci flanking K13 gene (31 900, 6 360, and 150 bp upstream and 11 000, 31 500, and 1 700 bp downstream) were amplified by PCR. Length of the PCR products was determined by a QIAxcel capillary electrophoresis instrument. The allele frequency and expected heterozygosity(He)were calculated by GenALEx software. Results Forty-six alleles were detected for the six microsatellite loci. The alleles of R539T mutant clustered on 286 bp(allele frequency 73%) and 278 bp (allele frequency 67%) for the locus of 6 360 bp upstream, and on 211 bp (allele frequency 55%) and 205 bp(allele frequency 44%) for 31 900 bp upstream. For the other two loci of 150 bp upstream and 1 700 bp downstream, the alleles mainly clustered on 195 bp and 209 bp. No alleles were found for the loci of 11 000 bp and 31 500 bp downstream. The haplotypes of the isolates in the three areas of Thailand(H2 type in 2 samples, and H5 and H6 in the rest samples) differed from that in Lazan(H1-H4 types). For the R539T mutant isolates from China-Burma border and Thailand, the mean He values were (0.49 ± 0.23) and (0.42 ± 0.33), respectively, while for the wild type isolates, the mean He values were (0.72 ± 0.12) and (0.67 ± 0.11), respectively. The expected heterozygosity was low at the loci closely adjacent to the K13 gene of the R539T mutant strains, with a He value of 0.17 at the 150 bp upstream locus and 0.3 at the 1 700 bp downstream locus. Conclusion P. falciparum parasites with R539T mutation of K13 gene are subjected to drug selection pressure in both China-Myanmar border and Thailand, and there is a significant allele difference at the upstream 6 360 bp and 31 900 bp loci.

Key words: Plasmodium falciparum, Microsatellite, K13 gene

中图分类号:

R382.312

陈学迪, 叶润, 潘卫庆. 中缅边境及泰国东南地区恶性疟原虫K13基因侧翼微卫星多态性研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 209-212.

Xue-di CHEN, Run YE, Wei-qing PAN. Polymorphisms of microsatellite loci flanking the K13 gene of Plasmodium falciparum in China-Myanmar border area and southeast areas of Thailand[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(3): 209-212.

图/表 3

参考文献 18

[1]	潘卫庆, 汤林华. 分子寄生虫学[M]. 上海: 上海科学技术出版社, 2004.
[2]	张苍林, 周红宁, 王剑, 等. 中缅边境地区恶性疟原虫对氯喹、哌喹、咯萘啶敏感性的体外测定[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(1): 41-44.
[3]	Heizmann P, Fernex M, Leimer R, et al. Plasmodium falciparum resistant to mefloquine plus sulfadoxine/pyrimethamine[J]. Lancet, 1986, 1(8477): 377.
[4]	Arnold K.Mefloquine and sulfadoxine/pyrimethamine resistant Plasmodium falciparum[J]. Lancet, 1986, 1(8473): 154.
[5]	Noedl H, Se Y, Schaecher K, et al. Evidence of artemisinin-resistant malaria in western Cambodia[J]. N Engl J Med, 2008, 359(24): 2619-2620.
[6]	Dondorp AM, Nosten F, Yi P, et al. Artemisinin resistance in Plasmodium falciparum malaria[J]. N Engl J Med, 2009, 361(5): 455-467.
[7]	张逸龙, 潘卫庆. 恶性疟原虫对青蒿素产生抗性的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(6): 418-424.
[8]	Ye R, Hu D, Zhang Y, et al. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border[J]. Sci Rep, 2016, 6(1): 20100.
[9]	Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[J]. Nature, 2014, 505(7481): 50-55.
[10]	Ménard D, Khim N, Beghain J, et al. A worldwide map of Plasmodium falciparum K13-Propeller polymorphisms[J]. N Engl J Med, 2016, 374(25): 2453-2464.
[11]	孙艾明, 董莹, 陈梦妮, 等. 云南省恶性疟原虫青蒿素耐药性相关基因K13 kelch结构域序列多态性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(4): 339-345.
[12]	Straimer J, Gnädig NF, Witkowski B, et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[J]. Science, 2015, 347(6220): 428-431.
[13]	Ghorbal M, Gorman M, Macpherson CR, et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system[J]. Nat Biotechnol, 2014, 32(8): 819-821.
[14]	Talundzic E, Okoth SA, Congpuong K, et al. Selection and spread of artemisinin-resistant alleles in Thailand prior to the global artemisinin resistance containment campaign[J]. PLoS Pathog, 2015, 11(4): e1004789.
[15]	Kunte R, Kunwar R.WHO Guidelines for the treatment of malaria[J]. Med J Armed Forces India, 2011, 67(4): 376-376.
[16]	赵绍敏, 王满元. 恶性疟原虫对青蒿素类药物产生耐药性的全球现状和基础研究[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(5): 380-384.
[17]	Mita T.Origins and spread of pfdhfr mutant alleles in Plasmodium falciparum[J]. Acta Trop, 2010, 114(3): 166-170.
[18]	Wootton JC, Feng X, Ferdig MT, et al. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum[J]. Nature, 2002, 418(6895): 320-323.

引物名称 Primer	正向Forward（5′→3′）	反向Reverse（5′→3′）
上游31 900 Forward 31 900	AATCAATCATGGAAATTAT	TTGTTCATTTAAGGGTAT-TT
上游6 360 Forward 6 360	AATTAGACAGGACTTGTTAATG	GGATATGTTATCCAAAATGCA
上游150 Forward 150	GATTCCCTATCATACGTCATAG	ACAAGGCGTAAATATTC-GTG
下游1 700 Reverse 1 700	ATTGGTAATTGATGATT-TGCTA	TACAGGAATATTGCTCA-CTCTA
下游11 000 Reverse 11 000	ATACTATGTTTTATTATCATGTCTT	ATAGGTACAAATGAAAA-AATGAT
下游31 500 Reverse 31 500	GTACAAATGAACCTCT-TCC	AATAATTCACCATGTCATGC