我国间日疟原虫分离株Duffy抗原结合蛋白基因多态性分析

发布时间：2018-04-17 01:14

本文选题：间日疟原虫 + PvDBP　；参考：《中国医科大学》2009年硕士论文

【摘要】： 目的探讨我国疟疾混合流行区间日疟原虫红内期候选抗原Duffy抗原结合蛋白(DBP)Ⅱ区基因多态性特点。方法从我国疟疾高发混合流行区云南省采集疟疾患者末梢血,制备血样干滤纸片,QIAamp DNA mini kit(QIAGEN,德国)试剂盒提取间日疟原虫基因组DNA。根据间日疟原虫标准株Sal-Ⅰ株DBPⅡ区为目的扩增片段设计特异性引物,以间日疟原虫基因组DNA为模板,采用高保真DNA聚合酶(KOD-plus聚合酶,Toyobo,日本),聚合酶链反应(PCR)扩增DBPⅡ区基因。对扩增所得片段纯化后,用美国ABIPRIME测序仪进行基因测序。Sal-Ⅰ标准株作为参照,利用BIOEDIT软件对所获得的基因序列进行排序(alignment)分析,检测基因突变位点。以DnaSP4.50.3(http://www.ub.es.dnasp/)和MEGA4.0软件评估序列多态性。结果 1、扩增了19个间日疟原虫DBPⅡ区(PvDBP RⅡ)基因序列600bp长度片段(碱基位点853～1452bp),编码200个氨基酸(密码子285～484)。 2、我国间日疟原虫云南分离株与Sal-Ⅰ标准株(Cent ral American株)比较,碱基水平共有13处发生突变,占2.17%(13/600),导致12个氨基酸位点发生改变,占6.00%(12/200),其中A1134→G位点为同义突变,未导致相应氨基酸发生改变;核苷酸改变共产生9种基因型,相应产生9种氨基酸型,以A1151G-G1169A-T1270A基因型(相应氨基酸型为D384G-R390H-L424I)为主(26.3%)。在碱基和氨基酸水平上未发现插入和缺失。 3、与国内外疟疾流行区PvDBP RⅡ氨基酸多态位点的观察比较显示,我国云南株PvDBP RⅡ氨基酸置换位点基本包含于国外报道的位点中,未发现地区特异性突变位点。 4、核苷酸序列多态性分析结果显示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)。讨论比较我国(云南、浙江和湖北)分离株的结果与南美、中东地区PvDBPRⅡ的基因多态性发现,我国分离株PvDBP RⅡ基因突变位点相对有限,提示我国分离株红内期候选抗原DBP蛋白功能相对保守。本研究结果显示,云南地区的PvDBP RⅡ的突变位点较浙江和湖北地区报道的位点,新发现3处突变位点,但发生频率均较低,这对分析我国不同流行区间PvDBP RⅡ基因多态性具有一定的提示意义。另外本研究还发现N417K和W437R位点呈关联出现,提示针对该靶位开发的多价疫苗也可能对我国流行区有效。分析我国云南分离株PvDBP RⅡ的基因多态性的原因,可能整体上受到自然选择的影响,呈正向选择趋势。我国云南分离株PvDBP RⅡ基因多态性突变位点的一些特点及影响多态性的可能因素分析,可为以PvDBP RⅡ为基础建立有效传播阻断疫苗的研究提供一定的线索。结论 1、与南美、中东、非洲等流行区相比,中国间日疟原虫混合流行区云南分离株PvDBP RⅡ的基因多态性相对有限。我国疟疾混合流行区云南与单一流行区湖北和浙江地区突变位点存在一定差异。 2、分析我国云南分离株PvDBP RⅡ的基因多态性的原因可能为整体上受到自然选择的影响,呈正向选择趋势。
[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.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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