论文标题:RV野毒株L基因C端3’-RACE扩增及序列分析
Amplification Carbo-tip of L Gene by 3"-RACE and Sequence Analysis of Rabies Virus
论文作者
论文导师 钱爱东,论文学位 硕士,论文专业 预防兽医学
论文单位 吉林农业大学,点击次数 245,论文页数 85页File Size3572K
2006-06-01论文网 http://www.lw23.com/lunwen_2522312/
RV;L terminal sequence; 3"-RACE; sequence analysis
狂犬病是人类最古老的疾病之一，也是当前世界上流行最广、危害最严重的人兽共患传染病。据WHO的最新统计，全球每年因狂犬病死亡的人数约为3.5～7万人，而造成家畜和其他动物死亡的损失更是无法估量。近年来随着分子生物学和免疫学的发展和应用，对狂犬病毒结构、基因序列及其主要功能以及发病和免疫机制等都有了新的认识和了解。本研究为了丰富我国狂犬病分子流行病学研究资料，对我国分离自不同动物的两个野毒株进行基因组部分序列的测定和分析。 本实验以吉林鹿源DRV8202株及河南鼠源野毒株MRV的L基因的C端为主要研究内容，用RT-PCR和3’-RACE方法对其进行了克隆和测序，将所测得的核苷酸序列和推导的氨基酸序列分成两个部分，一是L基因C端序列，另一个是L基因5’端的非编码区(NCS)，并与NCBI上公开发表的狂犬病毒其它毒株的相应片段进行分析比较。 结果表明DRV8202与MRV该段核苷酸序列及氨基酸序列同源性为91.3％和95.8％，两者均属于基因Ⅰ型。为了进一步分析两株狂犬病病毒基因及其产物的特点，在扩增片段序列末端从终止密码开始L基因5’端的非编码区序列，长54nt，将其与RV其他毒株相应序列进行比较。结果显示MRV与DRV8202株L基因5’末端核酸序列的同源性最低为64.8％。通过对NCS各种碱基的统计分析发现，所比较的这几株狂犬病毒末端G+C含量均高于38.89％，其中DRV8202株G+C含量为51.85％；MRV株为42.95％，而两株各种碱基组成含量相差较远。LP其氨基酸组成中以碱性氨基酸居多，蛋白质中的两个区域(第851～869和第1962～1980位)为疏水区，这两个区域可能参与与L蛋白同N和M1蛋白的相互作用，通过比对发现，在851～869区域DRV8202株病毒在856位上的Met突变为Val；在1962～1980区域MRV株在1971位的Ala突变为Ser，其他氨基酸均没有发生变异。
Rabies is a kind of zoonotic viral disease whose appearance has been notable throughout recorded history. It is the most popular and most severest infection disease in the world. WHO reported that there are 35,000-70,000 deaths each year worldwide. And the losses of domestic animal and other animal deaths are unable to estimate. In recent years along with molecular biology and immunology development and application, we know all aspects of rabies virus , such as virus structure, the gene sequence and its the main function and so on. In this study, the cDNA fragments of L gene 5" terminal of two rabies virus strains in Jilin and Henan were cloned and sequenced with method of RT-PCR and 3"-RACE . The nucleotides sequence and deduced amino acid sequence of these cDNA figments mentioned above were divided into two fractions. One is carbon part of L gene , the other is 5" terminal( NCS),and they were compared with other rabies-related virus strains published in NCBI previously.The result show that the homology of nucleotide sequence and amino acid sequence of carbon part in L gene among these two rabies virus strains is 91.3% and 95.8%, and these two strains belong to type of gene I. And comparison of the nucleotide sequence of L gene 5" terminal for deep research. It shows that the lowest homology among these rabies virus strains is DRV8202 and MRV, the homology is 64.8%. Statistics of each kind of basic group of NCS indicate that the contents of G+C% are all taller than 38.89%, contents of G+C% in DRV8202 is 51.85, and 42.95% in MRV. But each kind of basic group composition content is difference. Most of amino acid are basic amino acid in LP, two domains in LP are hydrophobic region (be situated at 851~869 and 1962-1980) ,they maybe participate in interaction of LP and N、 M1。 Through comparison, Met is mutated into Val at the site of 856 in the DRV8202 strain, Ala is mutated into Ser at the site of 1971 in the MRV strain, there are not mutations in other amino acids.
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