【作者】 李国辉；

【导师】 洪岸； 张玲；

【作者基本信息】 暨南大学 ， 遗传学， 2005， 硕士

【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(p<0.01);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,浓度更多还原

【Abstract】 Object: To investigate the mechanism of aggregating of rhbFGF protein in solution and the possibility of the aggregating degree decreasing by site-directed mutagenesis,we analysis the affecting factor of aggregating of rhbFGF protein in solution and compare the difference of rhbFGF（w） and rhbFGF（m） in aggregating;While screening some universial addictive to improve the solubility of hbFGF protein in solution and applying them to practice,so as to further better the stability of rhbFGF in the producing process.Method: By the solubility of hbFGF protein in solution and its mitogen in vitro as marker to indicate the difference of bFGF（w） and bFGF（m） ,which is in different salts and different pH system ,while comparing the difference on stability of different concentration protein and different concentration salts in solution; besides,we use non-reduced SDS-PAGE and HPLC method to analysis the component of supernant of bFGF solution and calculating the surface tension of the solution of adding different substance to explore the mechanism of the stability of rhbFGF protein in solution.Results: hbFGF（m） is 1. .Under the same conditions（pH7.0,NaC12.0M）,we can find that the aggregating degree of bFGF（w） is obviously higher than bFGF（m） at first stage and there are all some soluble aggregation in their supernant; under the same conditions,the solubility and biological activity of hbFGF（m） which is added two type sugars and two type polyols is increased with their concentration ,the largest solubility and biological activity of hbFGF（m） observed in 0.8M of sucrose and trehalose and in 6%（v/v） of glycerol and ethylene glycol,the protein concentration of hbFGF（m） is 1.145±0.0072mg/ml （sucrose）、 1.125±0.0056mg/ml （trehalose）、 0.831±0.0082 （ glycerol ） 、 1.053±0.0097 （ ethylene glycol ） , and biological activity is ED₅₀=0.48ng/ml and ED₅₀=0.58ng/ml and ED50=2.25ng/ml and ED50=1.85ng/mlrespectively.Conclusion:.Under the same conditions,the degree of aggregating and the speed of aggregation of hbFGF（w） is higher than hbFGF（m）. we find that aggregation depend on protein concentration,the higher bFGF in solution,the more polymer.Disulfide and hydrophobic interaction play marjor roles in the aggregating process of hbFGF in solution and it can decrease the aggregation of hbFGFwhen Cys sites is replaced by serines by site-directed mutagenesis.Trehalose and sucrose can prevent the aggregation of hbFGF（m） and increase its solubility in solution and the biological activity of hbFGF（m） in unit volume solution,so do glycerol and ethylene glycol. This will facilitate our further research in meliorating the stability of hbFGF in solution.更多还原