【作者】 吕新辉；

【导师】 毕振强； 纪绍忠； 阚飙；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2007， 硕士

【摘要】 目的：研究从我国大庆地区婴幼儿腹泻临床粪便标本中分离的一株A组人轮状病毒阳性毒株（命名为DQ-1株）VP7基因的基因分型，对DQ-1株VP7基因序列和糖蛋白VP7的蛋白质结构进行分析，并与已测序同血清型标准株和国内外地方株VP7基因序列进行序列比对。方法：通过RT-PCR技术扩增人A组轮状病毒DQ-1株VP7全长基因，并将RT-PCR产物连接到pMD18-T载体，转化大肠杆菌JM109感受态细胞，在含有氨苄青霉素的LB琼脂培养基上培养，形成单菌落后挑取白色菌落进行检测，从中筛选出两个PCR阳性菌落，并提取质粒，双酶切鉴定，最后利用通用引物进行测序。利用DNAStar软件对DQ-1株VP7基因测序序列与其他A组轮状病毒株VP7基因序列进行同源性比较；用RNAstructure4.4软件绘制DQ-1株VP7基因的二级结构；用Predictprotein软件对DQ-1株糖蛋白VP7蛋白结构进行分析。结果：DQ-1株VP7基因与同型的我国已测序G1型地方株T73、R96-197株基因序列同源性均为97.6％，与G1型标准株Wa株核苷酸序列同源性为92.9％，与G2血清型代表株DS-1株基因序列同源性为73.8％，与G3血清型代表株SA11株核甘酸序列同源性为75.6％，与G4血清型代表株J-4621株（78.1％）同源性也不高；其VP7基因mRNA可折叠多达20个发卡结构。DQ-1株VP7蛋白是由354个氨基酸组成的多肽，含有2个潜在的糖基化位点和多个磷酰化位点；DQ-1株氨基酸序列与T73、R96-197株同源性都达97.5％，与Wa株达94.6％，而与其它型DS-1株、SA11株和J-4621株分别仅为74.4％、79.4％和75.2％。因此，我们推定DQ-1株属于G1血清型。结论：DQ-1株VP7基因序列与已测序我国G1型地方株VP7基因序列几乎没有差别，而与G1型标准株VP7基因序列存在细微差异，这为我国人A组轮状病毒疫苗的引进和研制提供了分子生物学依据。目的：克隆中国不同地区四个B组成人腹泻轮状病毒株结构蛋白VP7基因，通过测序得到其核苷酸序列和氨基酸序列，比较与其它B组轮状病毒毒株基因和氨基酸序列同源性，从而进一步分析我国不同地区流行的B组成人腹泻轮状病毒毒株之间的系统进化关系。方法：参照文献设计引物，对中国不同地区流行的4株B组轮状病毒株进行RT-PCR，并将RT-PCR产物克隆到PCR^TMⅡ载体，转化E．Coli感受态细胞，提取转化菌质粒经限制性内切酶HamHⅠ和BglⅡ双酶切分析，从中筛选阳性重组质粒进行测序，测序结果采用DNAStar6.0进行多重序列比对分析，并与B组轮状病毒代表株和其它已测序地方株的核苷酸及氨基酸序列进行同源性比较，绘制系统遗传进化树。结果：本研究获得的4株B组成人腹泻轮状病毒株核苷酸序列同源性为97.3％～98.8％，氨基酸序列同源性为95.2％～99.3％之间；与B组成人腹泻轮状病毒代表株ADRV核苷酸序列同源性为98.2％～100％，氨基酸序列同源性为97.4％～100％；与国外B组成人腹泻轮状病毒株印度、孟加拉国株核苷酸序列同源性为90.1％～92.5％，氨基酸序列同源性为92.3％～95.6％。结论：研究表明所有B组成人腹泻轮状病毒毒株均属于同一血清型，这为成人腹泻轮状病毒的流行病学监测以及将来研制成人腹泻轮状病毒疫苗提供了分子生物学依据，同时本研究可以从分子病毒学理论上证明“病毒是以居群的形式存在的”和“病毒的居群内部存在一个’差异谱’”的说法。更多还原

【Abstract】 ObjectivesTo study strain DQ-1 VP7 gene variation of human group A rotavirus（ GARV） from da-qing district, we analyzed and compared its gene sequence and protein structure with standard strain and field strains of China which had been sequenced.MethodsFirstly we extract total RNA of DQ-1, the gene that encoded the structural protein VP7 gene of GARV was amplified from isolated strain DQ-1 by reverse transcription polymerase chain reaction（RT-PCR）.Then the products of RT-PCR were ligated with plasmid pMD18-T and transformed into E.coli competent cells JM109. Analyzed by PCR detection ,it was showed that the VP7 gene was cloned into pMD18-T and the recombinant plasmids were constructed by PCR detection. The VP7 gene of GARV strain DQ-1 was then sequenced. The identity to other GARV was analyzed by software DNAStar,the secondary structure of DQ-1 VP7 mRNA was predicted by software RNA structure 4.4 ,and the protein of VP7 was analyzed by Predictprotein.ResultsThe nucleotide sequence of DQ-1 VP7 gene showed high identities （more than 90 %） to that of other human GARV such as T73、R96-197 and Wa which belongs to G1 Genotype, while identities to that of G2 Genotype such as DS-1 and G3 Genotype such as SA11 and G4 Genotype such as J-4621 were lower than 80%.The secondary structure of DQ-1 VP7 mRNA was constructed by about 20 stem-loop structures.The polypeptide encoded by DQ-1 VP7 contains 354 amino acids,the protein contains two potential N-linked glycosylation sites and some phosphorylation sites. Compared with other GARV DQ-1 VP7 demonstrated 97.5% and 94.6% of amino acids similarity with T73 and Wa respectively,but only 74.4 %、79.4% and 75.2% with DS-1、SA11 and J-4621. So, we might draw the conclusion that strain DQ-1 belongs to G1 serotype.ConclusionsThe sequence analysis revealed that the VP7 gene of DQ-1 shared higher identities with field strains of G1 serotype ,but showed differences with standard strain ,these results might give a molecular biological evidence to the inducement and development of the GARV vaccine. ObjectivesTo compare gene and amino acid sequence identity to other group B rotavirus,and further analyse the Phylogenetic relationship of group B ADRV rotavirus that spreads different areas of china, we cloned and sequenced the structural protein VP7 gene of four strains human group B ADRV rotavirus in different areas of china.MethodsWe designed primers accordinging to the article, and extracted the RNA from four strains,then,VP7 genes were amplified by reverse transcription polymerase chain reaction（RT-PCR）. The products of RT-PCR were ligated with plasmid PCR^TMII and transformed into E.coli competent cells.Analyzed by PCR detection.it was showed that the VP7 gene was cloned into PCR^TMII and the recombinant plasmids were constructed. The VP7 genes of four strains were then sequenced. The identity to other group B rotavirus was analyzed by software DNAStar6.0, and thus the phylogenetic tree was established.Results The results show that the gene sequence identity between the four strains is 97.3%～98.8%, the ammo acid sequence identity is 95.2%～99.3%; the gene sequence identity to ADRV is 98.2%～100%, the amino acid sequence identity is 97.4%～100%; the gene sequence identity to foreign strains in India and Bangladesh is 90.1%～92.5%, the amino acid sequence identity is 92.3%～95.6%.ConclusionsThe sequence analysis revealed that all strains of human group B ADRV rotavirus belongs to the same serotype, these results can give a molecular biological evidence to the inducement of the group B ADRV rotavirus vaccine.They can also give a molecular virul evidence to the viewpoint that "Virus exists in the form of population"and "Inside the population of the Virus exists in a’variation spectrum’.更多还原