人呼吸道合胞病毒F蛋白的纯化与定量研究

发布时间：2018-01-07 09:21

本文关键词：人呼吸道合胞病毒F蛋白的纯化与定量研究　出处：《北京交通大学》2010年硕士论文　论文类型：学位论文

【摘要】： 人呼吸道合胞病毒(Human respiratory syncytial virus, RSV)是造成世界范围内婴幼儿下呼吸道病毒性感染最重要的病原。呼吸道合胞病毒融合蛋白(fusion protein, F)为病毒表面结构蛋白,在RSV A, B两种亚型中有很高的抗原保守性,是主要的保护性抗原,诱导中和抗体,被认为是很有潜力的候选疫苗。纯化RSV的F蛋白对诊断试剂和亚单位疫苗的研制具有重要意义。目前文献报道的F蛋白的纯化方法大多为免疫亲和层析和蛋白电泳法,这些方法不但蛋白产量少且会导致其抗原性的部分丧失,因此,为获得产率高、抗原性好的纯化的F蛋白,本论文提出了一种两步层析纯化RSV病毒包膜F蛋白的新方法并建立了F蛋白的夹心ELISA定量检测方法。由HEp-2细胞培养的RSV纯化天然的F蛋白,首先以RSV感染HEp-2细胞,待细胞出现病变(cytopathic effect, CPE)后,收获细胞及培养液,离心,取上清,PEG6000沉淀,获得的沉淀物经溶解后通过蔗糖密度梯度离心进一步纯化,纯化的病毒用含Triton X-100的裂解液溶解包膜蛋白,病毒蛋白粗提液依次经过离子交换层析和凝胶过滤层析对F蛋白进行纯化。经过两步层析可以得到高度纯化的F蛋白,且在纯化过程中保持了F蛋白的抗原性,纯化的F蛋白仍然能被一组单克隆抗体检测到。通过SDS-PAGE、Western Blot检测,其为140kD的同型二聚体。经此纯化方案,每10个T-75细胞培养瓶培养的病毒可以获得大约85μg的F蛋白。
[Abstract]:Human respiratory syncytial virus. RSVV is the most important pathogen of infantile lower respiratory tract virus infection worldwide. Respiratory syncytial virus fusion protein fusion protein. F) is a surface structural protein of virus, which is highly antigenically conservative in two subtypes of RSV A and B, and is the main protective antigen, which induces neutralizing antibodies. F protein purified from RSV is of great significance for the development of diagnostic reagent and subunit vaccine. Most of the methods reported in the literature at present are immunoaffinity chromatography and egg. White electrophoresis. These methods not only produce less protein, but also lead to partial loss of antigenicity. Therefore, in order to obtain high yield and good antigenicity of purified F protein. In this paper, a new method for purification of RSV virus envelope F protein by two step chromatography was proposed, and a sandwich ELISA quantitative detection method of F protein was established. The natural F was purified from RSV cultured by HEp-2 cells. Protein. First, the HEp-2 cells were infected with RSV. After the cytopathic effect (CPEs) appeared, the cells and culture medium were harvested, centrifuged, and supernatant was extracted. The obtained precipitate was further purified by sucrose density gradient centrifugation and the purified virus was dissolved in the lytic solution containing Triton X-100. F protein was purified by ion exchange chromatography and gel filtration chromatography, and the highly purified F protein was obtained by two steps chromatography, and the antigenicity of F protein was maintained in the process of purification. The purified F protein can still be detected by a group of monoclonal antibodies. It is a homotype dimer of 140 KD detected by SDS-PAGEG Blot. About 85 渭 g F protein could be obtained from every 10 T-75 cell culture bottles.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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