本文選題：間日瘧原蟲 + 乳酸脫氫酶�。� 參考：《安徽醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:分析江淮地區(qū)間日瘧原蟲乳酸脫氫酶基因(Plasmodium vivax lactate dehydrogenase. PvLDH)序列特征,以及是否存在多態(tài)性,研究其作為靶抗原在瘧疾診斷和新藥篩選中的意義及應(yīng)用前景。 方法:1、在安徽省蚌埠市區(qū)、五河縣、利辛縣等地采集間日瘧患者末梢血,制備厚薄血片及干血濾紙片。用QIAamp DNA mini kit(QIAGEN,德國)試劑盒提取干血濾紙片間日瘧原蟲基因組DNA(gDNA)39份,所有樣本經(jīng)染色鏡檢和PCR檢測鑒定均為單純間日瘧原蟲。2、根據(jù)GenBank數(shù)據(jù)庫中間日瘧原蟲Belem株LDH基因序列(NCBI No:DQ060151)設(shè)計引物,以提取的間日瘧原蟲基因組DNA為模板,PCR擴增LDH基因,產(chǎn)物經(jīng)瓊脂糖凝膠電泳純化回收后連接至pGEM-T質(zhì)粒,轉(zhuǎn)化E.coli DH5α菌株后挑取菌落進行PCR鑒定,將初步篩選的陽性克隆進行測序,最后通過Blast等方法進行序列的比對分析。 結(jié)果:39份患者血樣經(jīng)病原學(xué)染色鏡檢和PCR檢測均為單純間日瘧原蟲感染,未發(fā)現(xiàn)惡性瘧原蟲等其他瘧原蟲的單純或合并感染。擴增的PvLDH編碼基因克隆至T載體后測序顯示目的片段大小均為951bp,且39份樣本PvLDH核苷酸序列均無差異。通過BLAST方法對克隆的PvLDH基因序列進行比對分析,發(fā)現(xiàn)江淮地區(qū)間日瘧原蟲與Sal-I株、Belem株、薩爾瓦多株、EJEU60134株、MIMO612514株、ASAO6G29643株、ASAO6R09632株、MIAO61042株的LDH基因同源性高達99.7%-99.9%,只有1-3個堿基的差別;對PvLDH的氨基酸序列進行比對分析,結(jié)果發(fā)現(xiàn)江淮地區(qū)間日瘧原蟲與Sal-I株、Belem株等株間日瘧原蟲的LDH蛋白序列同源性為100%,與EJEU60134株、MIAO61251株的同源性為為99.7%,僅有1個氨基酸的差異。 結(jié)論:江淮地區(qū)間日瘧原蟲乳酸脫氫酶基因序列具有高度保守性,與Sal-I株、Belem株等基因序列比對分析LDH基因同源性達99.7%-99.9%,氨基酸序列比對分析基本為同義突變,僅與EJEU60134株和MIAO61251株有一個氨基酸的變化,該研究為間日瘧原蟲診斷試劑的研制提供了的一定的理論依據(jù)。
[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 偽 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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前8條

1 徐軍強;袁方玉;詹發(fā)先;黃光全;江永忠;陳麗;陳輝;;套式PCR檢測惡性瘧原蟲與間日瘧原蟲方法的建立及其應(yīng)用研究[J];公共衛(wèi)生與預(yù)防醫(yī)學(xué);2009年02期

2 翁屹,蘇成芝;重組質(zhì)粒DNA探針用于間日瘧診斷的研究[J];中國寄生蟲病防治雜志;1994年01期

3 王福勇,AllanSaul;惡性瘧原蟲乳酸脫氫酶的提純及其特異性鑒定 (英文)[J];中國寄生蟲病防治雜志;1994年01期

4 張龍興,柴建華,詹斌,馮曉平;DNA探針診斷間日瘧的進一步研究[J];中國寄生蟲學(xué)與寄生蟲病雜志;1993年02期

5 郭傳坤;黎學(xué)銘;李錦輝;毛瑋;林珍;杜進發(fā);黃天誼;;套式/多重PCR診斷瘧疾的敏感性、特異性和穩(wěn)定性初探[J];中國寄生蟲學(xué)與寄生蟲病雜志;2007年03期

6 周水森;王漪;房文;湯林華;;2007年全國瘧疾形勢[J];中國寄生蟲學(xué)與寄生蟲病雜志;2008年06期

7 江莉;王真瑜;馬曉疆;張小萍;蔡黎;;間日瘧原蟲和惡性瘧原蟲乳酸脫氫酶基因的序列和重組抗原表位分析[J];中國寄生蟲學(xué)與寄生蟲病雜志;2010年02期

8 方強;夏惠;王雪梅;齊文娟;常雪蓮;高琪;;間日瘧原蟲乳酸脫氫酶編碼區(qū)全長基因的克隆、序列分析及線性B細胞表位預(yù)測[J];中國人獸共患病學(xué)報;2010年06期

，

本文編號：1944148