重组人碱性成纤维细胞生长因子体外稳定性的研究

发布时间：2018-02-17 05:06

本文关键词： 碱性成纤维细胞生长因子聚合体突变添加剂　出处：《暨南大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:通过对重组人碱性成纤维细胞生长因子(recombinant human basic fibroblast growth factor,rhbFGF)体外聚合影响因素的研究;在野生型和突变型bFGF聚合行为的过程中,初步探讨聚合机理和氨基酸定点突变减少聚合的可能性;同时筛选常规使用的添加剂,并在生产工艺的部分环节检验小分子添加剂的使用效果,初步探讨小分子在蛋白质溶液中的稳定作用。方法:采用蛋白溶解度和促有丝分裂活性能力为指标,表征rhbFGF在不同的盐溶液、不同的pH体系以及不同蛋白浓度下的聚合反应,研究各因素对聚合反应的影响;对比野生型bFGF和突变型bFGF聚合程度的差异,采用电泳和高压液相色谱(HPLC)分析各溶液的组分以研究聚合反应的发生原因;在rhbFGF(m)溶液中添加小分子添加剂,研究小分子添加剂的类型和浓度对溶液中蛋白质的聚合程度的影响,利用溶液的表面张力测量数据对蛋白质稳定机制进行一些初步的探索。结果:通过聚合反应影响因素的研究,在不同目的的研究中可选择不同的缓冲溶液体系,通常情况下磷酸缓冲溶液(pH7.0)和NaCl是一个合适的选择。通过对bFGF(w)和bFGF(m)在体外形成聚合体进行比较和分析,发现bFGF(w)在溶液中聚合程度更大,活性损失较快,这两种类型的蛋白均发现可溶性聚合体存在。在相同的条件下,发现蔗糖、海藻糖浓度为0.8M时、甘油和乙二醇浓度(v/v)为6%时bFGF(m)可溶蛋白浓度和活性数值最高,所测得的蛋白浓度依次为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),与对照组比较均有显著差异(p0.01);添加有蔗糖、海藻糖、甘油和乙二醇的生物活性优于对照组,且在最佳作用浓度下测得: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)。结论:在相同体系(PBS10mmol/L、NaCl2.0M、pH7.0)中,溶液中bFGF(w)发生聚合程度明显高于bFGF(m),且发生聚合的程度与蛋白浓度呈依赖性,浓度
[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 卤0.0072 mg / ml (sucrose 1.125 卤0.0056 mg / ml) (trehalose 0.831 卤0.0082) (glycerol) 1.053 卤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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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