江淮地区间日疟原虫乳酸脱氢酶基因多态性研究

发布时间：2018-05-27 23:02

本文选题：间日疟原虫 + 乳酸脱氢酶　；参考：《安徽医科大学》2011年硕士论文

【摘要】：目的:分析江淮地区间日疟原虫乳酸脱氢酶基因(Plasmodium vivax lactate dehydrogenase. PvLDH)序列特征,以及是否存在多态性,研究其作为靶抗原在疟疾诊断和新药筛选中的意义及应用前景。方法:1、在安徽省蚌埠市区、五河县、利辛县等地采集间日疟患者末梢血,制备厚薄血片及干血滤纸片。用QIAamp DNA mini kit(QIAGEN,德国)试剂盒提取干血滤纸片间日疟原虫基因组DNA(gDNA)39份,所有样本经染色镜检和PCR检测鉴定均为单纯间日疟原虫。2、根据GenBank数据库中间日疟原虫Belem株LDH基因序列(NCBI No:DQ060151)设计引物,以提取的间日疟原虫基因组DNA为模板,PCR扩增LDH基因,产物经琼脂糖凝胶电泳纯化回收后连接至pGEM-T质粒,转化E.coli DH5α菌株后挑取菌落进行PCR鉴定,将初步筛选的阳性克隆进行测序,最后通过Blast等方法进行序列的比对分析。结果:39份患者血样经病原学染色镜检和PCR检测均为单纯间日疟原虫感染,未发现恶性疟原虫等其他疟原虫的单纯或合并感染。扩增的PvLDH编码基因克隆至T载体后测序显示目的片段大小均为951bp,且39份样本PvLDH核苷酸序列均无差异。通过BLAST方法对克隆的PvLDH基因序列进行比对分析,发现江淮地区间日疟原虫与Sal-I株、Belem株、萨尔瓦多株、EJEU60134株、MIMO612514株、ASAO6G29643株、ASAO6R09632株、MIAO61042株的LDH基因同源性高达99.7%-99.9%,只有1-3个碱基的差别;对PvLDH的氨基酸序列进行比对分析,结果发现江淮地区间日疟原虫与Sal-I株、Belem株等株间日疟原虫的LDH蛋白序列同源性为100%,与EJEU60134株、MIAO61251株的同源性为为99.7%,仅有1个氨基酸的差异。结论:江淮地区间日疟原虫乳酸脱氢酶基因序列具有高度保守性,与Sal-I株、Belem株等基因序列比对分析LDH基因同源性达99.7%-99.9%,氨基酸序列比对分析基本为同义突变,仅与EJEU60134株和MIAO61251株有一个氨基酸的变化,该研究为间日疟原虫诊断试剂的研制提供了的一定的理论依据。
[Abstract]:Objective: to analyze the lactate dehydrogenase gene of Plasmodium vivax lactate dehydrogenase. of Plasmodium vivax in Jianghuai area. The significance and application prospect of PVLDH as target antigen in malaria diagnosis and new drug screening were studied. Methods the peripheral blood of vivax malaria patients were collected in Bengbu, Wuhe and Lixin, Anhui Province. The genomic DNA(gDNA)39 of Plasmodium vivax was extracted from dry blood filter paper with QIAamp DNA mini kit QIAGEN (Germany). All samples were identified as Plasmodium vivax by staining microscopy and PCR detection. Primers were designed according to the LDH gene sequence of Belem strain of Plasmodium vivax in GenBank database. The LDH gene was amplified by PCR using the extracted genomic DNA of Plasmodium vivax as template. The product was purified and recovered by agarose gel electrophoresis and ligated into pGEM-T plasmid. After transformed into E.coli DH5 伪 strain, the colony was isolated and identified by PCR. The selected positive clones were sequenced. Finally, the sequence was compared and analyzed by Blast and other methods. Results the blood samples of 39 patients were detected by pathogenetic staining and PCR analysis. No single or combined infection of Plasmodium falciparum and other Plasmodium falciparum was found. The amplified PvLDH coding gene was cloned into T vector and sequenced. The size of the target fragment was 951 BP, and the nucleotide sequences of 39 samples were not different. The sequence of cloned PvLDH gene was analyzed by BLAST. It was found that the homology of LDH gene between Plasmodium vivax and Sal-I strain Belem, El Salvador strain EJEU60134, ASAO6G29643, ASAO6R09632 and ASAO6R09632 was 99.7- 99.9, only 1-3 bases. The amino acid sequence of PvLDH was compared. The results showed that the homology of LDH protein sequence between Plasmodium vivax and Sal-I strain Belem was 100, and 99.7 with EJEU60134 strain MIAO61251. The difference was only one amino acid. Conclusion: the lactate dehydrogenase gene sequence of Plasmodium vivax in Jianghuai area is highly conserved. The homology of LDH gene is 99.7- 99.9 and amino acid sequence alignment analysis is synonymous with Sal-I strain. There is only one amino acid change with EJEU60134 and MIAO61251 strains. This study provides a theoretical basis for the development of diagnostic reagent for Plasmodium vivax.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】