恶性疟原虫海南株氯喹抗性相关基因多态性及msp3等位基因分型研究

发布时间：2018-03-05 02:02

本文选题：恶性疟原虫　切入点：氯喹抗性　出处：《广东药学院》2007年硕士论文　论文类型：学位论文

【摘要】： 疟疾广泛流行至今仍无法得到很好控制的原因主要有以下几点:出现抗药性虫株和对杀虫剂产生抗性的媒介蚊虫,缺乏适当有效的疟疾疫苗等。疟疾流行导致大量人口死亡,能够引发严重的社会经济问题,几乎成为所有热带和亚热带国家的沉重负担。在曾广泛使用的抗疟药——氯喹对抗药性恶性疟失去其治疗效果后,这一问题更加严重。尽管氯喹抗性产生的具体机制还不十分清楚,但已有研究表明三种食物泡膜蛋白——Pgh1蛋白、一个约330kDa的蛋白以及PfCRT蛋白——可能在氯喹抗性产生的过程中发挥重要作用;而它们的编码基因——pfmdr1基因,cg2基因和pfcrt基因已被成功克隆,并且测定了序列。此外,一些研究认为具有不同基因型的恶性疟原虫可能具有不同的致病性、抗原性及药物敏感性,而且,某些分子标记在疟疾分子流行病学研究上具有重要价值。本论文主要包含两个方面:一是恶性疟原虫海南株氯喹抗性相关基因的多态性分析,二是对恶性疟原虫海南株裂殖子表面蛋白3进行等位基因分型。研究目的了解海南省恶性疟原虫pfcrt基因和pfmdr1基因特定位点的多态性,进一步分析其多态性同氯喹抗性的关系;对恶性疟原虫海南株进行msp3等位基因分型,了解海南省恶性疟原虫种群的构成特征。研究方法用WHO推荐的体外微量法测定海南省恶性疟原虫虫株对氯喹的敏感性。根据Genbank公布的恶性疟原虫pfcrt基因和pfmdr1基因序列设计引物,采用套式PCR分别扩增pfcrt基因编码第76位和第356位氨基酸的基因片段以及pfmdr1基因编码第86位、第1042位和第1246位氨基酸的基因片段。PCR产物分别用特定的限制性内切酶消化,分析其是否发生特定的点突变,并分析这些突变同氯喹抗性的关系。统计学分析抗性基因多态性同氯喹抗性的相关性。设计针对msp3基因两种不同等位基因型的特异引物,采用套式PCR方法分别扩增msp3基因不同等位基因型的目的基因片段。研究结果 42份采自海南省疟疾流行区恶性疟病人血样,经体外药物敏感性测试,22个为氯喹抗性株,20个为氯喹敏感性,总抗性率为52.38%。 pfmdr1基因:在全部42个样本中,第86位点均为野生型,没有发生突变。42个样本中有8个发生了D1246Y突变,其中除一例混合感染为氯喹敏感株外,其余均为抗性株。在对pfmdr1基因第1042位点的检测中有37个样本扩增得到目的基因片段,有10个发生了N1042D突变,其中9个为氯喹抗性株,1个为敏感株。 pfcrt基因:42个受试样本中,有30个(其中2个为混合感染)发生了K76T突变,其中18个为氯喹抗性株,12个为氯喹敏感株。第356位点未检测到突变,均为野生型。 msp3基因:42个样本中有36个扩增得到目的基因片段,属K1等位基因型的有25个(52.08%),属3D7等位基因型有23个(47.92%),其中同时扩增出两种等位基因片段的有12个,混合感染率为33.33%。结论海南省恶性疟原虫虫株pfcrt基因的K76T位点突变以及pfmdr1基因的N1042D位点突变和D1246Y位点突变同氯喹抗性具有统计学关系。而pfcrt基因的356位点和pfmdr1基因的86位点未发现具有多态性,与氯喹抗性不存在相关性。海南省恶性疟原虫虫株msp3等位基因型以K1型略占优势,3D7型较少,两种不同等位基因型虫株的混合感染率不高。
[Abstract]:Malaria prevalent is still not well controlled the main reasons are as follows: the emergence of drug-resistant strains and resistance to insecticides in mosquitoes, the lack of appropriate and effective malaria vaccine. The malaria epidemic led to a large number of deaths, can lead to serious social and economic problems, almost become a heavy burden on all tropical and subtropical countries in the widely used antimalarial drug chloroquine against falciparum malaria, losing its treatment effect, this problem is more serious. Although the specific mechanism of the emergence of chloroquine resistance is not very clear, but studies have shown that three kinds of food vacuole membrane protein, Pgh1 protein, plays an important role to produce about 330kDa the protein and PfCRT protein may be in chloroquine resistance; and the genes encoding pfmdr1 gene, CG2 gene and pfcrt gene has been cloned, and The sequence was determined. In addition, some studies with different gene types of Plasmodium falciparum may have different pathogenicity, and antigenicity and drug sensitivity, some molecular markers have important value in the study of molecular epidemiology of malaria. This paper mainly contains two aspects: one is the polymorphism analysis of Plasmodium falciparum isolates from Hainan chloroquine resistant genes the two is the Hainan strain of Plasmodium falciparum merozoite surface protein 3 alleles.
research objective
Objective to understand polymorphism of pfcrt and pfmdr1 gene loci in Plasmodium falciparum in Hainan Province, further analyze the relationship between polymorphism and chloroquine resistance, and identify MSP3 genotypes of Plasmodium falciparum Hainan strain, so as to understand the constitution characteristics of Plasmodium falciparum population in Hainan province.
research method
Determination of sensitivity to chloroquine in Hainan province of P. falciparum isolates in vitro microdilution method recommended by WHO. The primers were designed according to pfcrt gene of Plasmodium falciparum pfmdr1 gene and the published sequences of Genbank gene fragments were amplified by nested PCR pfcrt gene encoding seventy-sixth and 356th amino acid and pfmdr1 gene encoding the eighty-sixth gene fragments,.PCR products 1042nd and 1246th amino acids respectively with specific restriction enzyme digestion analysis, whether the occurrence of specific point mutations and mutation analysis of these relations with chloroquine resistance. Statistical analysis of resistance gene polymorphism associated with chloroquine resistance.
We designed specific primers for two different alleles of MSP3 gene, and amplified the target gene fragments of different alleles of MSP3 gene by nested PCR.
Research results
A total of 42 blood samples from falciparum malaria patients in malaria endemic area of Hainan province were tested by in vitro drug sensitivity test, 22 were chloroquine resistant plants, 20 were chloroquine sensitive, and the total resistance rate was 52.38%..
Pfmdr1 gene: in all 42 samples, eighty-sixth loci were wild type, no mutation in.42 samples in 8 of the D1246Y mutation, which in addition to a case of mixed infection of chloroquine sensitive strains and resistant strains. The rest are in the detection of pfmdr1 gene 1042nd loci in 37 samples amplified get the objective gene fragment, 10 had N1042D mutations, 9 of which were chloroquine resistant strains, 1 sensitive strains.
Pfcrt gene: 30 out of 42 tested samples (2 of them were mixed infection) had K76T mutation, 18 of them were chloroquine resistant strains, 12 were chloroquine sensitive strains. 356th loci did not detect mutations, all of them were wild type.
MSP3 gene: 36 out of 42 samples were amplified and the target gene fragments were obtained. 25 alleles (52.08%) belonged to the K1 allele, 23 alleles (47.92%) belonged to the 3D7 allele, among which two alleles were 12, and the mixed infection rate was 33.33%..
conclusion
N1042D site K76T site in Hainan province of P. falciparum isolates pfcrt gene mutation and pfmdr1 gene mutation and D1246Y mutation has significant relations with chloroquine resistance. 356 loci and pfmdr1 gene pfcrt gene 86 loci were found with polymorphism, there was no correlation with chloroquine resistance.
Hainan province of P. falciparum isolates MSP3 allele with K1 type a slight advantage, 3D7 less, a mixture of two different allelic type strains, the infection rate is not high.

【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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