绿色荧光蛋白表达的重组巨细胞病毒的构建及抗巨细胞病毒药物筛选
发布时间：2018-12-17 03:16
【摘要】：人巨细胞病毒,属于疱疹病毒亚科,是自然界中普遍存在的病原体,大部分的巨细胞病毒感染是无临床症状的,但是可以在机体中长期潜伏存在,引起免疫缺陷者严重的疾病甚至死亡。近几年来,随着器官移植,免疫抑制治疗,HIV感染者的增加,巨细胞病毒感染的影响越来越受到人们的重视。而且,巨细胞病毒感染也是引起新生儿出生缺陷的主要病原体之一。由于该病毒结构的复杂性,至今对于其确切的致病机理及机体对该病毒感染的保护性免疫反应仍知之甚少,治疗比较困难,有效药物并不多。近年来抗病毒的药物有了很大的发展,抗巨细胞病毒的常用药物的有更昔洛韦,西多福韦,磷甲酸等,但是存在口服药物生物效价低,药效差,一定的毒性以及耐药毒株的问题。随着医药基础理论的不断前进,基因工程的不断发展,通过广大医药工作者的不懈努力,新的抗病毒药不断出现。我校药学院研究开发了一系列的抗巨细胞病毒的药物-噻吩并噻二嗪及其衍生物,与本课题组合作对其进行抗巨细胞病毒药物筛选。目前国内学者常用的抗病毒药物的筛选方法是细胞病变效应法(CPE法),该方法需要的实验周期比较长,比较复杂,但是结果是肯定的。其他国外学者有用DNA杂交技术和流式细胞术检测抗原的方法,但是受到实验室条件和技术的限制。本课题利用分子生物学方法,构建绿色荧光蛋白的巨细胞病毒,建立了一种快速有效的抗巨细胞病毒的药物筛选方法,通过检测绿色荧光蛋白的表达来判断药物的作用,并拟将该重组病毒进一步用于巨细胞病毒发病机制的研究。 首先,将PHM673质粒转化入大肠杆菌DH5α,筛选阳性克隆,扩增纯化后得到大量质粒,酶切鉴定,将质粒直线化,用脂质体转染法转入原代培养的人胚肺成纤维细胞,巨细胞病毒Towne标准株感染转染后细胞,G418筛选,空斑法纯化,PCR鉴定,获得携带绿色荧光蛋白的重组巨细胞病毒。将HCMV-Towne
[Abstract]:Human cytomegalovirus, belonging to the herpesvirus subfamily, is a common pathogen in nature. Most cytomegalovirus infections are asymptomatic, but can be latent in the body for a long time. It causes serious illness and even death in people with immune deficiency. In recent years, with organ transplantation, immunosuppressive therapy and the increase of HIV infection, the effect of cytomegalovirus infection has been paid more and more attention. Moreover, cytomegalovirus infection is also one of the main pathogens causing birth defects in newborns. Due to the complexity of the structure of the virus, little is known about its exact pathogenetic mechanism and the protective immune response of the body to the virus infection, the treatment is difficult and the effective drugs are not many. In recent years, antiviral drugs have been greatly developed. Common anti-cytomegalovirus drugs include ganciclovir, cidofovir, phosphoric acid, etc. Certain toxicity and the problem of drug resistant strains. With the development of the basic theory of medicine and the development of genetic engineering, new antiviral drugs have been emerging through the unremitting efforts of the vast number of medical workers. A series of anticytomegalovirus drugs, thiophenothiadiazine and its derivatives, have been developed in our institute of pharmacy, and their anticytomegalovirus drugs have been screened in cooperation with our team. At present, cytopathic effect method (CPE) is the common method for screening antiviral drugs, which requires a long and complex experimental period, but the results are positive. Other foreign scholars use DNA hybridization and flow cytometry to detect antigens, but they are limited by laboratory conditions and techniques. In this paper, we construct cytomegalovirus of green fluorescent protein by molecular biology, and establish a rapid and effective drug screening method to detect the expression of green fluorescent protein. The recombinant virus will be further used to study the pathogenesis of cytomegalovirus. Firstly, the PHM673 plasmid was transformed into Escherichia coli DH5 伪, positive clones were screened, a large number of plasmids were obtained after amplification and purification, the plasmids were identified by restriction endonuclease digestion, the plasmids were linearized and transferred into primary cultured human embryonic lung fibroblasts by liposome transfection. The recombinant cytomegalovirus carrying green fluorescent protein was obtained by G418 screening, plaque purification and PCR identification after infection with the standard cytomegalovirus Towne strain. HCMV-Towne
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
本文编号：2383558
[Abstract]:Human cytomegalovirus, belonging to the herpesvirus subfamily, is a common pathogen in nature. Most cytomegalovirus infections are asymptomatic, but can be latent in the body for a long time. It causes serious illness and even death in people with immune deficiency. In recent years, with organ transplantation, immunosuppressive therapy and the increase of HIV infection, the effect of cytomegalovirus infection has been paid more and more attention. Moreover, cytomegalovirus infection is also one of the main pathogens causing birth defects in newborns. Due to the complexity of the structure of the virus, little is known about its exact pathogenetic mechanism and the protective immune response of the body to the virus infection, the treatment is difficult and the effective drugs are not many. In recent years, antiviral drugs have been greatly developed. Common anti-cytomegalovirus drugs include ganciclovir, cidofovir, phosphoric acid, etc. Certain toxicity and the problem of drug resistant strains. With the development of the basic theory of medicine and the development of genetic engineering, new antiviral drugs have been emerging through the unremitting efforts of the vast number of medical workers. A series of anticytomegalovirus drugs, thiophenothiadiazine and its derivatives, have been developed in our institute of pharmacy, and their anticytomegalovirus drugs have been screened in cooperation with our team. At present, cytopathic effect method (CPE) is the common method for screening antiviral drugs, which requires a long and complex experimental period, but the results are positive. Other foreign scholars use DNA hybridization and flow cytometry to detect antigens, but they are limited by laboratory conditions and techniques. In this paper, we construct cytomegalovirus of green fluorescent protein by molecular biology, and establish a rapid and effective drug screening method to detect the expression of green fluorescent protein. The recombinant virus will be further used to study the pathogenesis of cytomegalovirus. Firstly, the PHM673 plasmid was transformed into Escherichia coli DH5 伪, positive clones were screened, a large number of plasmids were obtained after amplification and purification, the plasmids were identified by restriction endonuclease digestion, the plasmids were linearized and transferred into primary cultured human embryonic lung fibroblasts by liposome transfection. The recombinant cytomegalovirus carrying green fluorescent protein was obtained by G418 screening, plaque purification and PCR identification after infection with the standard cytomegalovirus Towne strain. HCMV-Towne
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
【引证文献】
相关期刊论文 前1条
1 刘程彬;宋宏锐;杨鹏;;绿色荧光蛋白在药物发现研究中的应用进展[J];沈阳药科大学学报;2013年04期
，本文编号：2383558
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2383558.html
最近更新
教材专著
