HCMV临床病毒株的分离及其特定基因结构分析和表达研究

发布时间：2018-04-21 08:50

本文选题：人巨细胞病毒 + 临床病毒株　；参考：《暨南大学》2006年博士论文

【摘要】：目的：1．分离广州地区围产新生儿感染的人巨细胞病毒(HumanCytomegalovirus，HCMV)临床病毒株；2．研究这些低传代HCMV临床病毒株基因组中的特有基因UL136～UL148的结构，分析其结构多态性与HCMV先天性感染致病的关系；3．研究HCMV临床毒株特有基因的表达情况，探讨这些基因编码产物的功能，揭示它们在先天性HCMV感染的致病机制中可能的作用。试图通过这些研究为临床低传代HCMV临床致病机制的研究奠定基础。方法：1．从广州地区先天性感染HCMV的新生儿尿液中分离野生型HCMV病毒，获取病毒DNA和RNA；2．根据GenBank公布的HCMV低传代毒株Toledo的UL136-UL148等13个基因全序列及有关文献，应用PCR技术扩增这些特有基因片段；3．应用基因克隆技术构建这些特有基因的重组质粒；4．对重组体进行测序；5．应用生物信息学方法分析这些基因结构、及其编码蛋白质的多态性；6．应用RT-PCR技术检测上述基因mRNA的表达情况；7．并分析其编码产物的基本性质和特征。结果：1．成功分离获得4株HCMV临床低传代病毒株：HCMV D3、D2、D52、C30；2．以HCMV D3临床毒株为研究主体，应用PCR技术成功扩增出UL136～UL148特有区域的全部13个基因，同时扩增其他临床毒株的相应基因，，获得上述毒株的13个特有基因的总共32个全基因序列；3．克隆测序，序列呈报GenBank，收录序列号为：DQ180354～DQ180391；4．对临床毒株的这些基因及其编码的蛋白质进行同源性比较分析，发现大部分基因较为保守，具有相对稳定的序列，同时又具有较为丰富的多态性；5．从C30野生毒株中能检测到UL137、UL142、UL144、UL145和UL148等5个基因存在mRNA水平的表达。结论：1．广州地区围产新生儿感染HCMV的临床毒株基因组中，存在着实验病毒株所缺失的UL136～UL148等13个独特基因结构，并且具有较为丰富的多态性。2．本研究初步证实了其中UL137、UL142、UL144、UL145、UL148等5
[Abstract]:Purpose 1. Human cytomegalovirus (HCMV) clinical virus strain of human cytomegalovirus (HCMV) was isolated from perinatal neonates in Guangzhou area. To study the structure of the unique gene UL136~UL148 in the genome of these low passage HCMV clinical virus strains and to analyze the relationship between its structural polymorphism and the pathogenesis of congenital HCMV infection. To study the expression of genes specific to clinical strains of HCMV, to explore the function of these gene coding products, and to reveal their possible role in the pathogenesis of congenital HCMV infection. Through these studies, we try to lay a foundation for the study of clinical pathogenesis of low passage HCMV. Method 1: 1. Wild-type HCMV virus was isolated from urine of neonates with congenital HCMV infection in Guangzhou area, and virus DNA and RNA2 were obtained. According to the whole sequence of 13 genes of HCMV low passage strain Toledo and related literature published by GenBank, the specific gene fragment was amplified by PCR technique. The recombinant plasmids of these unique genes were constructed by gene cloning technique. The recombinant was sequenced. Bioinformatics was used to analyze the structure of these genes and the polymorphism of their encoded proteins. RT-PCR technique was used to detect the expression of mRNA. The basic properties and characteristics of the coded products are analyzed. The result is 1: 1. Four HCMV clinical low passage virus strains were successfully isolated and obtained. Using HCMV D3 clinical strain as the main research subject, all 13 genes in the specific region of UL136~UL148 were successfully amplified by PCR technique, and the corresponding genes of other clinical strains were amplified simultaneously. A total of 32 complete gene sequences of 13 unique genes of the above mentioned strains were obtained. Cloning and sequencing, the sequence was reported to GenBank.The serial number was:: DQ180354 / DQ180391. The homology analysis of these genes and their encoded proteins in clinical strains showed that most of the genes were conserved, had relatively stable sequences, and had abundant polymorphisms at the same time. The expression of mRNA was detected in 5 genes of UL137, UL142U144U144U145 and UL148 from C30 wild strain. Conclusion 1. In the clinical genome of perinatal neonates infected with HCMV, there are 13 unique gene structures, such as UL136~UL148, which are missing from the experimental virus strains, and there are abundant polymorphisms. 2. This study has preliminarily confirmed that UL137FU, UL142FU, UL144FU, UL145U, et al.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R373

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