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  本文關(guān)鍵詞： 堿性成纖維細胞生長因子 聚合體 突變 添加劑　出處：《暨南大學》2005年碩士論文　論文類型：學位論文

【摘要】：目的:通過對重組人堿性成纖維細胞生長因子(recombinant human basic fibroblast growth factor,rhbFGF)體外聚合影響因素的研究;在野生型和突變型bFGF聚合行為的過程中,初步探討聚合機理和氨基酸定點突變減少聚合的可能性;同時篩選常規(guī)使用的添加劑,并在生產(chǎn)工藝的部分環(huán)節(jié)檢驗小分子添加劑的使用效果,初步探討小分子在蛋白質(zhì)溶液中的穩(wěn)定作用。 方法:采用蛋白溶解度和促有絲分裂活性能力為指標,表征rhbFGF在不同的鹽溶液、不同的pH體系以及不同蛋白濃度下的聚合反應,研究各因素對聚合反應的影響;對比野生型bFGF和突變型bFGF聚合程度的差異,采用電泳和高壓液相色譜(HPLC)分析各溶液的組分以研究聚合反應的發(fā)生原因;在rhbFGF(m)溶液中添加小分子添加劑,研究小分子添加劑的類型和濃度對溶液中蛋白質(zhì)的聚合程度的影響,利用溶液的表面張力測量數(shù)據(jù)對蛋白質(zhì)穩(wěn)定機制進行一些初步的探索。 結(jié)果:通過聚合反應影響因素的研究,在不同目的的研究中可選擇不同的緩沖溶液體系,通常情況下磷酸緩沖溶液(pH7.0)和NaCl是一個合適的選擇。通過對bFGF(w)和bFGF(m)在體外形成聚合體進行比較和分析,發(fā)現(xiàn)bFGF(w)在溶液中聚合程度更大,活性損失較快,這兩種類型的蛋白均發(fā)現(xiàn)可溶性聚合體存在。在相同的條件下,發(fā)現(xiàn)蔗糖、海藻糖濃度為0.8M時、甘油和乙二醇濃度(v/v)為6%時bFGF(m)可溶蛋白濃度和活性數(shù)值最高,所測得的蛋白濃度依次為1.145±0.0072mg/ml(蔗糖)、1.125±0.0056mg/ml(海藻糖)、0.831±0.0082(甘油)、1.053±0.0097(乙二醇),與對照組比較均有顯著差異(p0.01);添加有蔗糖、海藻糖、甘油和乙二醇的生物活性優(yōu)于對照組,且在最佳作用濃度下測得:ED50=0.48ng/ml(蔗糖)、ED50=0.58ng/ml(海藻糖);ED50=2.25ng/ml(甘油)、ED50=1.85ng/ml(乙二醇)。 結(jié)論:在相同體系(PBS10mmol/L、NaCl2.0M、pH7.0)中,溶液中bFGF(w)發(fā)生聚合程度明顯高于bFGF(m),且發(fā)生聚合的程度與蛋白濃度呈依賴性,濃度
[Abstract]:Objective: to study the influencing factors of recombinant human basic fibroblast growth factor (human basic fibroblast growth factor-rhbFGFs) in vitro, and to investigate the polymerization behavior of wild type and mutant type bFGF. The mechanism of polymerization and the possibility of reducing polymerization by amino acid site-directed mutation were discussed preliminarily. At the same time, conventional additives were screened, and the effects of small molecular additives were tested in some parts of the production process. The stabilization of small molecules in protein solution was preliminarily discussed. Methods: protein solubility and mitogenic activity were used to characterize the polymerization of rhbFGF in different salt solution, different pH system and different protein concentration, and the effects of various factors on the polymerization were studied. Comparing the degree of polymerization of wild type bFGF and mutant type bFGF, the components of each solution were analyzed by electrophoresis and high pressure liquid chromatography (HPLC) to study the reason of polymerization, and the small molecule additive was added to the solution of rhbFGFM. The effects of the type and concentration of small molecular additives on the degree of protein polymerization in solution were studied. The mechanism of protein stabilization was preliminarily explored by measuring the surface tension of the solution. Results: through the study of the influencing factors of polymerization reaction, different buffer solution systems could be selected in the study of different purposes. Through the comparison and analysis of the formation of polymer in vitro, it was found that the polymerization degree of bFGFW) in solution was greater and the loss of activity was faster. Soluble polymers were found in both types of proteins. Under the same conditions, the highest concentrations and activities of soluble proteins were found when the concentration of sucrose and trehalose was 0.8m, and the concentration of glycerol and ethylene glycol was 6. The protein concentration measured was 1.145 鹵0.0072 mg / ml (sucrose 1.125 鹵0.0056 mg / ml) (trehalose 0.831 鹵0.0082) (glycerol) 1.053 鹵0.0097 (ethylene glycol), and the biological activity of sucrose, trehalose, glycerol and ethylene glycol was better than that of control group. And at the optimum concentration, 1% ED50 + 0.48 ng / ml (Sucrose / ED50 + 0.58 ng / ml) / ml (ED50 + 2.25 ng / ml) / ml (ED50 + 1.85 ng / ml) (ethylene glycol). Conclusion: in the same system, the degree of polymerization in solution is obviously higher than that in the solution, and the degree of polymerization is dependent on the concentration of protein.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R341

【引證文獻】

相關(guān)碩士學位論文 前2條

1 許媛媛;膠原海綿的改性及其作為活性因子載體的研究[D];中國人民解放軍軍事醫(yī)學科學院;2008年

2 馮程程;堿性成纖維生長因子鼻腔噴霧劑的研制及腦內(nèi)遞藥特性研究[D];復旦大學;2011年

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本文編號：1517285