發(fā)布時間：2018-12-17 03:16

【摘要】：人巨細(xì)胞病毒,屬于皰疹病毒亞科,是自然界中普遍存在的病原體,大部分的巨細(xì)胞病毒感染是無臨床癥狀的,但是可以在機(jī)體中長期潛伏存在,引起免疫缺陷者嚴(yán)重的疾病甚至死亡。近幾年來,隨著器官移植,免疫抑制治療,HIV感染者的增加,巨細(xì)胞病毒感染的影響越來越受到人們的重視。而且,巨細(xì)胞病毒感染也是引起新生兒出生缺陷的主要病原體之一。由于該病毒結(jié)構(gòu)的復(fù)雜性,至今對于其確切的致病機(jī)理及機(jī)體對該病毒感染的保護(hù)性免疫反應(yīng)仍知之甚少,治療比較困難,有效藥物并不多。近年來抗病毒的藥物有了很大的發(fā)展,抗巨細(xì)胞病毒的常用藥物的有更昔洛韋,西多福韋,磷甲酸等,但是存在口服藥物生物效價低,藥效差,一定的毒性以及耐藥毒株的問題。隨著醫(yī)藥基礎(chǔ)理論的不斷前進(jìn),基因工程的不斷發(fā)展,通過廣大醫(yī)藥工作者的不懈努力,新的抗病毒藥不斷出現(xiàn)。我校藥學(xué)院研究開發(fā)了一系列的抗巨細(xì)胞病毒的藥物-噻吩并噻二嗪及其衍生物,與本課題組合作對其進(jìn)行抗巨細(xì)胞病毒藥物篩選。目前國內(nèi)學(xué)者常用的抗病毒藥物的篩選方法是細(xì)胞病變效應(yīng)法(CPE法),該方法需要的實(shí)驗(yàn)周期比較長,比較復(fù)雜,但是結(jié)果是肯定的。其他國外學(xué)者有用DNA雜交技術(shù)和流式細(xì)胞術(shù)檢測抗原的方法,但是受到實(shí)驗(yàn)室條件和技術(shù)的限制。本課題利用分子生物學(xué)方法,構(gòu)建綠色熒光蛋白的巨細(xì)胞病毒,建立了一種快速有效的抗巨細(xì)胞病毒的藥物篩選方法,通過檢測綠色熒光蛋白的表達(dá)來判斷藥物的作用,并擬將該重組病毒進(jìn)一步用于巨細(xì)胞病毒發(fā)病機(jī)制的研究。 首先,將PHM673質(zhì)粒轉(zhuǎn)化入大腸桿菌DH5α,篩選陽性克隆,擴(kuò)增純化后得到大量質(zhì)粒,酶切鑒定,將質(zhì)粒直線化,用脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)入原代培養(yǎng)的人胚肺成纖維細(xì)胞,巨細(xì)胞病毒Towne標(biāo)準(zhǔn)株感染轉(zhuǎn)染后細(xì)胞,G418篩選,空斑法純化,PCR鑒定,獲得攜帶綠色熒光蛋白的重組巨細(xì)胞病毒。將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 偽, 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
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R373

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉程彬;宋宏銳;楊鵬;;綠色熒光蛋白在藥物發(fā)現(xiàn)研究中的應(yīng)用進(jìn)展[J];沈陽藥科大學(xué)學(xué)報;2013年04期

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本文編號：2383558