山东省间日疟原虫MSP-1和CSP等位基因型及同源性分析
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摘要
目的分析山东省间日疟原虫MSP-1和CSP基因类型及其同源性,为病例溯源提供科学依据。方法采集2011年山东省报告的12例间日疟患者血样,提取疟原虫基因组DNA;分别根据间日疟原虫MSP-1和CSP基因序列设计引物,进行巢式PCR扩增、酶切、测序、序列比对及同源性分析。结果 12份间日疟患者血样MSP-1基因全部出现470bp扩增条带以及350、120 bp酶切片段,均为Sal-1型;MSP-1进化树分析显示,9份省内感染者样品序列同属一个分枝,1份印度感染者样品序列与印度分离株位于同一分枝。12份间日疟患者样品CSP基因均包含GDRA(D/A)GQPA序列,为PV-Ⅰ型,其中10份省内感染者和1份广东感染者样品CSP基因出现560~840 bp和150~230 bp两种扩增条带,为PV-Ⅰ型温带族,1份在印度感染者样品CSP基因仅出现560~840 bp条带,为PV-Ⅰ型热带族。CSP进化树表明,10份省内感染者及1份广东感染者样品序列同属一个分枝,1份在印度感染者样品序列与印度和印度尼西亚分离株位于同一分枝。结论山东省本地感染间日疟原虫MSP-1基因型均为Sal-1型,CSP基因型均为PV-Ⅰ型温带族,本地虫株具有较强的基因同源性。
Objective To analyze the genotypes and homology of MSP-1 and CSP gene of Plasmodium vivax in Shandong Province,so as to provide the evidence for case traceability. Methods A total of 12 blood samples were collected from P. vivaxinfected cases in Shandong Province in 2011. Parasite genomic DNA was extracted. Primers were designed according to MSP-1and CSP gene sequences of P. vivax. Then Nested PCR,enzyme digestion,sequencing and sequence alignment,and homologous analysis were performed. Results The MSP-1 gene of all the 12 samples from P. vivax-infected cases were detected with a 470 bp PCR amplification band,and 350 bp and 120 bp enzyme digestion fragments,which were identified as type Sal-1. An analysis of phylogenetic tree of MSP-1 gene showed that the sequences of 9 indigenous case samples in Shandong Province were located in the same branch,one case sample infected from India was located in the same branch with India strains. All the 12 P.vivax-infected samples covered GDRA(D/A)GQPA sequences in CSP gene,which were identified as type PV-Ⅰ. Of the CSP gene among 12 P. vivax-infected samples,10 samples of indigenous case in Shandong Province and one sample of the case infected in Guangdong Province were detected with both 560-840 bp and 150-230 bp PCR amplification bands,which were identified as temperate zone family strain of type PV-Ⅰ. However,one sample from the case infected in India was detected only with a 560-840 bp band,which was identified as tropical zone family strain of PV-Ⅰ. An analysis of phylogenetic tree of CSP gene showed that the sequences of 10 samples from the indigenous cases in Shandong Province and one sample from the case infected in Guangdong Province were located in the same branch,one sample from the case infected in India was located in the same branch with India and Indonesia strains. Conclusion Of all the indigenous isolates in Shandong Province,MSP-1 gene is genotyped type Sal-1,CSP gene is genotyped temperate zone family strain of type PV-Ⅰ,with a high homology found among the indigenous isolates.
Objective To analyze the genotypes and homology of MSP-1 and CSP gene of Plasmodium vivax in Shandong Province,so as to provide the evidence for case traceability. Methods A total of 12 blood samples were collected from P. vivaxinfected cases in Shandong Province in 2011. Parasite genomic DNA was extracted. Primers were designed according to MSP-1and CSP gene sequences of P. vivax. Then Nested PCR,enzyme digestion,sequencing and sequence alignment,and homologous analysis were performed. Results The MSP-1 gene of all the 12 samples from P. vivax-infected cases were detected with a 470 bp PCR amplification band,and 350 bp and 120 bp enzyme digestion fragments,which were identified as type Sal-1. An analysis of phylogenetic tree of MSP-1 gene showed that the sequences of 9 indigenous case samples in Shandong Province were located in the same branch,one case sample infected from India was located in the same branch with India strains. All the 12 P.vivax-infected samples covered GDRA(D/A)GQPA sequences in CSP gene,which were identified as type PV-Ⅰ. Of the CSP gene among 12 P. vivax-infected samples,10 samples of indigenous case in Shandong Province and one sample of the case infected in Guangdong Province were detected with both 560-840 bp and 150-230 bp PCR amplification bands,which were identified as temperate zone family strain of type PV-Ⅰ. However,one sample from the case infected in India was detected only with a 560-840 bp band,which was identified as tropical zone family strain of PV-Ⅰ. An analysis of phylogenetic tree of CSP gene showed that the sequences of 10 samples from the indigenous cases in Shandong Province and one sample from the case infected in Guangdong Province were located in the same branch,one sample from the case infected in India was located in the same branch with India and Indonesia strains. Conclusion Of all the indigenous isolates in Shandong Province,MSP-1 gene is genotyped type Sal-1,CSP gene is genotyped temperate zone family strain of type PV-Ⅰ,with a high homology found among the indigenous isolates.
引文
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[8]张山鹰,陆惠民,许龙善,等.我国不同疟区间日疟原虫裂殖子表面蛋白1(Pv MSP-1)基因多态性研究[J].中国人兽共患病杂志,2004,20(1):26-30.
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[10]黄天谊,黄亚铭,王小力,等.我国间日疟原虫基因型种群结构及其地理分布[J].中国寄生虫学与寄生虫病杂志,2001,19(5):260-264.
[11]徐贵全,张再兴,李娜,等.中缅边境间日疟原虫环子孢子蛋白基因分型[J].中国寄生虫学与寄生虫病杂志,2010,28(4):265-267.
[12]Imwong M,Pukrittayakamee S,Grüner AC,et al.Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1[J].Malar J,2005,4(1):20.
[13]卜秀芹,王用斌,赵长磊,等.2010年山东省疟疾综合防治措施效果[J].中国血吸虫病防治杂志,2012,24(1):116,118.
[14]刘新,李桂萍,傅斌,等.山东省输入性疟疾的监测与防治[J].中国寄生虫病防治杂志,2002,15(3):Ⅱ-Ⅲ.
[15]Kolakovich KA,Ssengoba A,Wojcik K,et al.Plasmodium vivax:Favored gene frequencies of the merozoite surface protein-1 and the multiplicity of infection in a malaria endemic region[J].Exp Parasitol,1996,83(1):11-18.
[16]Lim CS,Kim SH,Kwon S,et al.Analysis of Plasmodium vivax merozoite surface protein-1 gene sequences from resurgent korean isolates[J].Am J Trop Med Hyg,2000,62(2):261-265.
[17]Putaporntip C,Jongwutiwes S,Sakihama N,et al.Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus[J].Proc Natl Acad Sci U S A,2002,99(25):16348-16353.
[18]李雨春,王善青,胡锡敏,等.海南省消除疟疾后期间日疟原虫裂殖子表面蛋白1基因(MSP-1)多态性检测[J].中国人兽共患病学报,2014,30(2):219-222.
[19]耿英芝,毛玲玲,腾聪,等.辽宁丹东间日疟原虫裂殖子表面蛋1等位基因型分析[J].中国寄生虫学与寄生虫病杂志,2012,30(3):242-244.
[20]刘颖,许汴利,杨成运,等.间日疟原虫环子孢子蛋白(Pv CSP)基因多态性研究进展[J].中国病原生物学杂志,2016,11(1):90-93.
[21]黎学铭,郭传坤,李锦辉,等.我国南方五省间日疟原虫环子孢子蛋白基因型与疟疾防治效果关系的探讨[J].中国寄生虫学与寄生虫病杂志,2005,23(5):274-276,282.
[22]耿英芝,田疆,腾聪,等.辽宁省间日疟原虫环子孢子蛋白基因型分析[J].中国病原生物学杂志,2012,7(5):344-346.
[23]刘颖,周瑞敏,陈伟奇,等.间日疟原虫环子孢子蛋白PCRRFLP多态性分析[J].中国寄生虫学与寄生虫病杂志,2013,31(6):483-485.
[2]Cui L,Escalante AA,Imwong M,et al.The genetic diversity of Plasmodium vivax populations[J].Trends Parasitol,2003,19(5):220-226.
[3]Del Portillo HA,Longacre S,Khouri E,et al.Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species[J].Proc Natl Acad Sci U S A,1991,88(9):4030-4034.
[4]Gibson HL,Tucker JE,Kaslow DC,et al.Structure and expression of the gene for Pv200,a major blood-stage surface antigen of Plasmodium vivax[J].Mol Biochem Parasitol,1992,50(2):325-333.
[5]黄演婷,卢雪梅,金小宝,等.疟原虫环子孢子蛋白的研究进展[J].中国寄生虫学与寄生虫病杂志,2012,30(3):238-242.
[6]Rosenberg R,Wirtz RA,Lanar D,et al.Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax[J].Science,1989,245(4921):973-976.
[7]黄天谊,王小力,黎学铭,等.间日疟原虫环子孢子蛋白基因型的鉴别研究[J].中国寄生虫学与寄生虫病杂志,2000,18(5):272-276.
[8]张山鹰,陆惠民,许龙善,等.我国不同疟区间日疟原虫裂殖子表面蛋白1(Pv MSP-1)基因多态性研究[J].中国人兽共患病杂志,2004,20(1):26-30.
[9]张山鹰,许龙善,陆惠民,等.间日疟原虫裂殖子表面蛋白的等位基因型检测[J].中国寄生虫学与寄生虫病杂志,2004,22(2):86-89.
[10]黄天谊,黄亚铭,王小力,等.我国间日疟原虫基因型种群结构及其地理分布[J].中国寄生虫学与寄生虫病杂志,2001,19(5):260-264.
[11]徐贵全,张再兴,李娜,等.中缅边境间日疟原虫环子孢子蛋白基因分型[J].中国寄生虫学与寄生虫病杂志,2010,28(4):265-267.
[12]Imwong M,Pukrittayakamee S,Grüner AC,et al.Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1[J].Malar J,2005,4(1):20.
[13]卜秀芹,王用斌,赵长磊,等.2010年山东省疟疾综合防治措施效果[J].中国血吸虫病防治杂志,2012,24(1):116,118.
[14]刘新,李桂萍,傅斌,等.山东省输入性疟疾的监测与防治[J].中国寄生虫病防治杂志,2002,15(3):Ⅱ-Ⅲ.
[15]Kolakovich KA,Ssengoba A,Wojcik K,et al.Plasmodium vivax:Favored gene frequencies of the merozoite surface protein-1 and the multiplicity of infection in a malaria endemic region[J].Exp Parasitol,1996,83(1):11-18.
[16]Lim CS,Kim SH,Kwon S,et al.Analysis of Plasmodium vivax merozoite surface protein-1 gene sequences from resurgent korean isolates[J].Am J Trop Med Hyg,2000,62(2):261-265.
[17]Putaporntip C,Jongwutiwes S,Sakihama N,et al.Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus[J].Proc Natl Acad Sci U S A,2002,99(25):16348-16353.
[18]李雨春,王善青,胡锡敏,等.海南省消除疟疾后期间日疟原虫裂殖子表面蛋白1基因(MSP-1)多态性检测[J].中国人兽共患病学报,2014,30(2):219-222.
[19]耿英芝,毛玲玲,腾聪,等.辽宁丹东间日疟原虫裂殖子表面蛋1等位基因型分析[J].中国寄生虫学与寄生虫病杂志,2012,30(3):242-244.
[20]刘颖,许汴利,杨成运,等.间日疟原虫环子孢子蛋白(Pv CSP)基因多态性研究进展[J].中国病原生物学杂志,2016,11(1):90-93.
[21]黎学铭,郭传坤,李锦辉,等.我国南方五省间日疟原虫环子孢子蛋白基因型与疟疾防治效果关系的探讨[J].中国寄生虫学与寄生虫病杂志,2005,23(5):274-276,282.
[22]耿英芝,田疆,腾聪,等.辽宁省间日疟原虫环子孢子蛋白基因型分析[J].中国病原生物学杂志,2012,7(5):344-346.
[23]刘颖,周瑞敏,陈伟奇,等.间日疟原虫环子孢子蛋白PCRRFLP多态性分析[J].中国寄生虫学与寄生虫病杂志,2013,31(6):483-485.