本文選題：間日瘧原蟲 + PvDBP�。� 參考：《中國醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的 探討我國瘧疾混合流行區(qū)間日瘧原蟲紅內(nèi)期候選抗原Duffy抗原結(jié)合蛋白(DBP)Ⅱ區(qū)基因多態(tài)性特點(diǎn)。 方法 從我國瘧疾高發(fā)混合流行區(qū)云南省采集瘧疾患者末梢血,制備血樣干濾紙片,QIAamp DNA mini kit(QIAGEN,德國)試劑盒提取間日瘧原蟲基因組DNA。根據(jù)間日瘧原蟲標(biāo)準(zhǔn)株Sal-Ⅰ株DBPⅡ區(qū)為目的擴(kuò)增片段設(shè)計(jì)特異性引物,以間日瘧原蟲基因組DNA為模板,采用高保真DNA聚合酶(KOD-plus聚合酶,Toyobo,日本),聚合酶鏈反應(yīng)(PCR)擴(kuò)增DBPⅡ區(qū)基因。對(duì)擴(kuò)增所得片段純化后,用美國ABIPRIME測(cè)序儀進(jìn)行基因測(cè)序。Sal-Ⅰ標(biāo)準(zhǔn)株作為參照,利用BIOEDIT軟件對(duì)所獲得的基因序列進(jìn)行排序(alignment)分析,檢測(cè)基因突變位點(diǎn)。以DnaSP4.50.3(http://www.ub.es.dnasp/)和MEGA4.0軟件評(píng)估序列多態(tài)性。 結(jié)果 1、擴(kuò)增了19個(gè)間日瘧原蟲DBPⅡ區(qū)(PvDBP RⅡ)基因序列600bp長度片段(堿基位點(diǎn)853～1452bp),編碼200個(gè)氨基酸(密碼子285～484)。 2、我國間日瘧原蟲云南分離株與Sal-Ⅰ標(biāo)準(zhǔn)株(Cent ral American株)比較,堿基水平共有13處發(fā)生突變,占2.17%(13/600),導(dǎo)致12個(gè)氨基酸位點(diǎn)發(fā)生改變,占6.00%(12/200),其中A1134→G位點(diǎn)為同義突變,未導(dǎo)致相應(yīng)氨基酸發(fā)生改變;核苷酸改變共產(chǎn)生9種基因型,相應(yīng)產(chǎn)生9種氨基酸型,以A1151G-G1169A-T1270A基因型(相應(yīng)氨基酸型為D384G-R390H-L424I)為主(26.3%)。在堿基和氨基酸水平上未發(fā)現(xiàn)插入和缺失。 3、與國內(nèi)外瘧疾流行區(qū)PvDBP RⅡ氨基酸多態(tài)位點(diǎn)的觀察比較顯示,我國云南株P(guān)vDBP RⅡ氨基酸置換位點(diǎn)基本包含于國外報(bào)道的位點(diǎn)中,未發(fā)現(xiàn)地區(qū)特異性突變位點(diǎn)。 4、核苷酸序列多態(tài)性分析結(jié)果顯示π=0.0091(SE:0.0028);dn/ds=6.168(P＜0.05);Tajima's D=1.21360(P＞0.10);Fu and Li's D*=1.48091(P＜0.05),F*=1.62640(P＜0.05)。 討論 比較我國(云南、浙江和湖北)分離株的結(jié)果與南美、中東地區(qū)PvDBPRⅡ的基因多態(tài)性發(fā)現(xiàn),我國分離株P(guān)vDBP RⅡ基因突變位點(diǎn)相對(duì)有限,提示我國分離株紅內(nèi)期候選抗原DBP蛋白功能相對(duì)保守。 本研究結(jié)果顯示,云南地區(qū)的PvDBP RⅡ的突變位點(diǎn)較浙江和湖北地區(qū)報(bào)道的位點(diǎn),新發(fā)現(xiàn)3處突變位點(diǎn),但發(fā)生頻率均較低,這對(duì)分析我國不同流行區(qū)間PvDBP RⅡ基因多態(tài)性具有一定的提示意義。另外本研究還發(fā)現(xiàn)N417K和W437R位點(diǎn)呈關(guān)聯(lián)出現(xiàn),提示針對(duì)該靶位開發(fā)的多價(jià)疫苗也可能對(duì)我國流行區(qū)有效。 分析我國云南分離株P(guān)vDBP RⅡ的基因多態(tài)性的原因,可能整體上受到自然選擇的影響,呈正向選擇趨勢(shì)。我國云南分離株P(guān)vDBP RⅡ基因多態(tài)性突變位點(diǎn)的一些特點(diǎn)及影響多態(tài)性的可能因素分析,可為以PvDBP RⅡ?yàn)榛A(chǔ)建立有效傳播阻斷疫苗的研究提供一定的線索。 結(jié)論 1、與南美、中東、非洲等流行區(qū)相比,中國間日瘧原蟲混合流行區(qū)云南分離株P(guān)vDBP RⅡ的基因多態(tài)性相對(duì)有限。我國瘧疾混合流行區(qū)云南與單一流行區(qū)湖北和浙江地區(qū)突變位點(diǎn)存在一定差異。 2、分析我國云南分離株P(guān)vDBP RⅡ的基因多態(tài)性的原因可能為整體上受到自然選擇的影響,呈正向選擇趨勢(shì)。
[Abstract]:objective
The polymorphisms of the Duffy antigen binding protein (DBP) II region gene of the erythroid candidate antigen of Plasmodium vivax in the mixed epidemic area of malaria in China were investigated.
Method
Mixed epidemic area in Yunnan province collecting blood from China malaria malaria, preparation of dry blood filter paper, QIAamp DNA Mini Kit (QIAGEN, Germany) kit to extract genomic DNA. of Plasmodium vivax according to standard strains of Sal- strain in DBP II region for the purpose of amplified fragment specific primers to Plasmodium vivax genome DNA as a template, using high fidelity DNA polymerase (KOD-plus polymerase, Toyobo, Japan), polymerase chain reaction (PCR) amplification of gene DBP II. The purified PCR amplified fragment, using the United States ABIPRIME sequencing of gene sequencing of.Sal- of standard strain as a reference, to sort the gene sequence obtained by BIOEDIT software (alignment) analysis, detection of gene mutation loci. In DnaSP4.50.3 (http://www.ub.es.dnasp/) and MEGA4.0 in the assessment of sequence polymorphism.
Result
1, we amplified 19 Plasmodium vivax DBP II region (PvDBP R II) gene sequences 600bp length fragment (base site 853 to 1452bp), encoding 200 amino acids (codon 285~484).
2, China's Yunnan vivax isolates and standard strains (Cent ral Sal- I American strain), a total of 13 base mutations, accounted for 2.17% (13/600), resulting in 12 amino acid changes, which accounted for 6% (12/200), A1134, G loci were synonymous mutation, did not lead to the corresponding amino acid the change of nucleotide change; a total of 9 genotypes, 9 kinds of amino acid type corresponding to A1151G-G1169A-T1270A genotype (the corresponding amino acid type D384G-R390H-L424I). (26.3%). Insertions and deletions were found in nucleotide and amino acid levels.
3, compared with the PvDBP R II amino acid polymorphic loci in malaria endemic area at home and abroad, the amino acid substitution sites of PvDBP R II in Yunnan strain were basically included in the reported loci abroad, and no loci specific mutation sites were found.
4, nucleotide sequence polymorphism analysis showed that PI =0.0091 (SE:0.0028); dn/ds=6.168 (P < 0.05); Tajima's D=1.21360 (P > 0.10); Fu and Li's D*=1.48091 (and < 0.05), and ((< 0.05)).
discuss
Comparing the results of isolates from China (Yunnan, Zhejiang and Hubei) and PvDBPR II gene polymorphisms in South America and the Middle East, we found that the PvDBP R II gene mutation sites in China were relatively limited, suggesting that the DBP protein in Chinese erythrocytic candidate antigens is relatively conservative.
The results of this study show that mutations compared with Zhejiang and Hubei area reported site of PvDBP R II in Yunnan area, and found 3 mutations, but the frequency was low, which has certain significance to prompt analysis of polymorphic interval PvDBP R II gene in different fashion. The study also found that the N417K addition and W437R loci were related, suggesting that the target for development of multivalent vaccines may also be on China's endemic areas.
Analysis of the causes of China's Yunnan isolate PvDBP R II gene polymorphism may, subject to the overall impact of natural selection, positive selection trend. China's Yunnan isolates PvDBP R II gene polymorphism mutation site some characteristics and the impact of the polymorphism analysis of possible factors, can be applied to PvDBP based R II effective transmission blocking vaccine research provide some clues.
conclusion
1, compared with the epidemic areas such as South America, Middle East and Africa, the polymorphism of PvDBP R II gene in the mixed epidemic area of Plasmodium vivax in China is relatively limited. There are some differences in the mutation sites between Yunnan and the single epidemic area Hubei and Zhejiang in the mixed malaria endemic area of China.
2, the analysis of the genetic polymorphisms of PvDBP R II in Yunnan, China, may be influenced by natural selection as a whole, showing a positive trend of selection.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.1

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本文編號(hào)：1761409