本文選題：惡性瘧原蟲(chóng)　切入點(diǎn)：氯喹抗性　出處：《廣東藥學(xué)院》2007年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 瘧疾廣泛流行至今仍無(wú)法得到很好控制的原因主要有以下幾點(diǎn):出現(xiàn)抗藥性蟲(chóng)株和對(duì)殺蟲(chóng)劑產(chǎn)生抗性的媒介蚊蟲(chóng),缺乏適當(dāng)有效的瘧疾疫苗等。瘧疾流行導(dǎo)致大量人口死亡,能夠引發(fā)嚴(yán)重的社會(huì)經(jīng)濟(jì)問(wèn)題,幾乎成為所有熱帶和亞熱帶國(guó)家的沉重負(fù)擔(dān)。在曾廣泛使用的抗瘧藥——氯喹對(duì)抗藥性惡性瘧失去其治療效果后,這一問(wèn)題更加嚴(yán)重。盡管氯喹抗性產(chǎn)生的具體機(jī)制還不十分清楚,但已有研究表明三種食物泡膜蛋白——Pgh1蛋白、一個(gè)約330kDa的蛋白以及PfCRT蛋白——可能在氯喹抗性產(chǎn)生的過(guò)程中發(fā)揮重要作用;而它們的編碼基因——pfmdr1基因,cg2基因和pfcrt基因已被成功克隆,并且測(cè)定了序列。此外,一些研究認(rèn)為具有不同基因型的惡性瘧原蟲(chóng)可能具有不同的致病性、抗原性及藥物敏感性,而且,某些分子標(biāo)記在瘧疾分子流行病學(xué)研究上具有重要價(jià)值。本論文主要包含兩個(gè)方面:一是惡性瘧原蟲(chóng)海南株氯喹抗性相關(guān)基因的多態(tài)性分析,二是對(duì)惡性瘧原蟲(chóng)海南株裂殖子表面蛋白3進(jìn)行等位基因分型。 研究目的 了解海南省惡性瘧原蟲(chóng)pfcrt基因和pfmdr1基因特定位點(diǎn)的多態(tài)性,進(jìn)一步分析其多態(tài)性同氯喹抗性的關(guān)系;對(duì)惡性瘧原蟲(chóng)海南株進(jìn)行msp3等位基因分型,了解海南省惡性瘧原蟲(chóng)種群的構(gòu)成特征。 研究方法 用WHO推薦的體外微量法測(cè)定海南省惡性瘧原蟲(chóng)蟲(chóng)株對(duì)氯喹的敏感性。根據(jù)Genbank公布的惡性瘧原蟲(chóng)pfcrt基因和pfmdr1基因序列設(shè)計(jì)引物,采用套式PCR分別擴(kuò)增pfcrt基因編碼第76位和第356位氨基酸的基因片段以及pfmdr1基因編碼第86位、第1042位和第1246位氨基酸的基因片段。PCR產(chǎn)物分別用特定的限制性內(nèi)切酶消化,分析其是否發(fā)生特定的點(diǎn)突變,并分析這些突變同氯喹抗性的關(guān)系。統(tǒng)計(jì)學(xué)分析抗性基因多態(tài)性同氯喹抗性的相關(guān)性。 設(shè)計(jì)針對(duì)msp3基因兩種不同等位基因型的特異引物,采用套式PCR方法分別擴(kuò)增msp3基因不同等位基因型的目的基因片段。 研究結(jié)果 42份采自海南省瘧疾流行區(qū)惡性瘧病人血樣,經(jīng)體外藥物敏感性測(cè)試,22個(gè)為氯喹抗性株,20個(gè)為氯喹敏感性,總抗性率為52.38%。 pfmdr1基因:在全部42個(gè)樣本中,第86位點(diǎn)均為野生型,沒(méi)有發(fā)生突變。42個(gè)樣本中有8個(gè)發(fā)生了D1246Y突變,其中除一例混合感染為氯喹敏感株外,其余均為抗性株。在對(duì)pfmdr1基因第1042位點(diǎn)的檢測(cè)中有37個(gè)樣本擴(kuò)增得到目的基因片段,有10個(gè)發(fā)生了N1042D突變,其中9個(gè)為氯喹抗性株,1個(gè)為敏感株。 pfcrt基因:42個(gè)受試樣本中,有30個(gè)(其中2個(gè)為混合感染)發(fā)生了K76T突變,其中18個(gè)為氯喹抗性株,12個(gè)為氯喹敏感株。第356位點(diǎn)未檢測(cè)到突變,均為野生型。 msp3基因:42個(gè)樣本中有36個(gè)擴(kuò)增得到目的基因片段,屬K1等位基因型的有25個(gè)(52.08%),屬3D7等位基因型有23個(gè)(47.92%),其中同時(shí)擴(kuò)增出兩種等位基因片段的有12個(gè),混合感染率為33.33%。 結(jié)論 海南省惡性瘧原蟲(chóng)蟲(chóng)株pfcrt基因的K76T位點(diǎn)突變以及pfmdr1基因的N1042D位點(diǎn)突變和D1246Y位點(diǎn)突變同氯喹抗性具有統(tǒng)計(jì)學(xué)關(guān)系。而pfcrt基因的356位點(diǎn)和pfmdr1基因的86位點(diǎn)未發(fā)現(xiàn)具有多態(tài)性,與氯喹抗性不存在相關(guān)性。 海南省惡性瘧原蟲(chóng)蟲(chóng)株msp3等位基因型以K1型略占優(yōu)勢(shì),3D7型較少,兩種不同等位基因型蟲(chóng)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.

【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R383

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本文編號(hào)：1568245