【作者】 周亚滨；

【导师】 于修平；

【作者基本信息】 山东大学 ， 病原生物学， 2005， 硕士

【摘要】 人巨细胞病毒(HCMV)是胎儿宫内感染和围产期感染的重要病原,以及因免疫抑制剂、器官和骨髓移植、艾滋病等所致免疫低下患者患严重感染的重要病原体之一,妊娠妇女感染HCMV可通过胎盘传染给胎儿,导致胎儿发育迟缓、畸形、低体重儿、黄疸、皮疹等。HCMV也是器官移植失败和艾滋病病人并发感染死亡的主要原因,对非免疫缺陷者常导致长期隐性感染或出现非特异症状,甚至引起感染细胞的转化、畸变或癌变,同时也与多种肿瘤的发生相关,如宫颈癌、前列腺癌、结肠癌及卡波济肉瘤。 HCMV主要糖蛋白gB广泛表达在病毒颗粒及感染细胞表面,具有被病毒中和抗体识别的抗原表位,能特异性的诱导机体的免疫反应,因此被认为是HCMV疫苗的重要候选蛋白,制备巨细胞病毒gB疫苗,对于巨细胞病毒感染所诱发的疾病可起到积极的防治作用。 目前许多抗病毒药物对HCMV感染引起的相关疾病有较好的疗效,但对需要长期维持治疗的病人常产生耐药现象,且均存在不同程度的毒性作用,给临床治疗带来困难。因此,研制新型抗HCMV药物,对于HCMV感染引起的相关疾病的治疗是非常必须的。 研究内容: 一、HCMV主要包膜糖蛋白gB疫苗的构建及免疫原性检测 1.利用分子生物学技术构建构建人巨细胞病毒(HCMV)包膜糖蛋白gB真核基因表达载体pCMV.gB。 2.初步评价其作为核酸疫苗在小鼠体内的免疫效果 通过皮下接种免疫小鼠,测量小鼠的体温、体重等一般情况,利用LDH释放试验检测免疫小鼠的NK细胞活性,MTT法检测T细胞增殖活性、IL-2活性,ELISA方法检测小鼠的抗HCMV IgM、IgG抗体及IL-2、IL-4含量。 实验结果显示:成功构建gB表达载体pCMV.gB,用其免疫小鼠后,更多还原

【Abstract】 Human cytomegalovirus is the important etiology infected in fetal intrauterine infection and perinatal stage. And it is also the one important pathogen of some serious disease which caused by immuno-inhibitor used, organs and bone marrow transplantation or AIDS. Many research show that HUMV is more related to some tumor, such as cervical carcinoma, prostate carcinoma. Colon cancer and so on.HCMV gB can express on the surface of virion and infected cell, the Ag epitope which recognesed by specific Ab can induce the immune reaction , so it is the important protein for prevent the disease which caused by HCMV.And now, researches showed that many anti-viral drugs have a better curative effect on some disease caused by HCMV. However, in some long-term treatment patiens, the drug resistance is easy to occure. And we also found that the drug which used to treatment the infection caused by virus have some virulence to body. So ,it is important to develop a new anti-viral drug to therapy the infection of HCMV. I. Constructing HCMV gB gene expression vector as DNA vaccine and detecting its immunityObjective to construct human cytomegalovirus glycoprotein B expression vector pCMV.gB and to evaluate its immunological effects as DNA vaccine in the mouse in-vivo model.Methods we constructed HCMV pCMV.gB expression vector by inserted PCR production gB gene to the plasmid pcDNA3. BALB/C mice were immunized by pCMV.gB by hypodermic injection. Body temperature and weight was measuredevery two weeks .NK cell activity was tested by LDH release test. MTT method was used to detect T cell proliferation activity and IL-2 biological activity. The serum IgM and IgG levels of HCMV were detected using ELISA method. The concentrations of IL-2 and IL-4 in sera and supernatant of spleen cells were detected using ELISA.Results The HCMV DNA vaccine expression vectors pCMV.gB were successfully constructed. The common conditions of mice were well after being immunized. There was no significant difference of weight and body temperature between the immunized mice and the corresponding control. T cell proliferation activity and NK cell activity significantly increased, and specified IgM and IgG for HCMV were induced. The concentrations of IL-2 and IL-4 and activity of IL-2 in supernatant of spleen cells were increased.ConclusionHCMV DNA vaccine of pCMV.gB induced immunological reaction by this approach. II. Study the mechanism of the new anti-viral drug1. To observe the virulence of the drug2. To observe the control effect of the drug3.To examine the mRNA of IE gene and L gene of HCMV by RT-PCR. Conclusion: The study showed that the drug is safety to the culture cell and caninhibit the replication of HCMV.更多还原