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  本文關(guān)鍵詞：人呼吸道合胞病毒F蛋白的純化與定量研究　出處：《北京交通大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人呼吸道合胞病毒 純化 F蛋白 離子交換層析 凝膠過濾層析 夾心ELISA

【摘要】： 人呼吸道合胞病毒(Human respiratory syncytial virus, RSV)是造成世界范圍內(nèi)嬰幼兒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)為病毒表面結(jié)構(gòu)蛋白,在RSV A, B兩種亞型中有很高的抗原保守性,是主要的保護性抗原,誘導(dǎo)中和抗體,被認為是很有潛力的候選疫苗。純化RSV的F蛋白對診斷試劑和亞單位疫苗的研制具有重要意義。目前文獻報道的F蛋白的純化方法大多為免疫親和層析和蛋白電泳法,這些方法不但蛋白產(chǎn)量少且會導(dǎo)致其抗原性的部分喪失,因此,為獲得產(chǎn)率高、抗原性好的純化的F蛋白,本論文提出了一種兩步層析純化RSV病毒包膜F蛋白的新方法并建立了F蛋白的夾心ELISA定量檢測方法。由HEp-2細胞培養(yǎng)的RSV純化天然的F蛋白,首先以RSV感染HEp-2細胞,待細胞出現(xiàn)病變(cytopathic effect, CPE)后,收獲細胞及培養(yǎng)液,離心,取上清,PEG6000沉淀,獲得的沉淀物經(jīng)溶解后通過蔗糖密度梯度離心進一步純化,純化的病毒用含Triton X-100的裂解液溶解包膜蛋白,病毒蛋白粗提液依次經(jīng)過離子交換層析和凝膠過濾層析對F蛋白進行純化。經(jīng)過兩步層析可以得到高度純化的F蛋白,且在純化過程中保持了F蛋白的抗原性,純化的F蛋白仍然能被一組單克隆抗體檢測到。通過SDS-PAGE、Western Blot檢測,其為140kD的同型二聚體。經(jīng)此純化方案,每10個T-75細胞培養(yǎng)瓶培養(yǎng)的病毒可以獲得大約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.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R373

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本文編號：1391946