【摘要】 对重组毕赤酵母表达人乳头瘤病毒病毒样颗粒发酵条件进行了优化。在全合成摇瓶培养基的基础上,考察了诱导过程中培养液pH、温度、铵离子、油酸对人乳头瘤病毒病毒样颗粒发酵的影响。研究表明发酵过程中单体蛋白16L1的表达条件与组装条件不同:pH=4.5最适于单体L1表达,而pH=3.5则最适于VLPs组装;28℃利于单体L1表达,而30℃则促进VLPs组装;发酵指数生长期诱导时铵离子浓度为0.26~0.30mol/L,利于菌体生长、单体L1表达与组装;生长稳定期时铵离子浓度为0.34~0.42mol/L,最有利于单体的合成与组装。进一步研究发现,诱导初期添加油酸,会抑制单体L1表达,而在诱导中期添加φ=5%油酸则能够极大地促进L1自组装。毕赤酵母发酵过程中胞内单体L1表达与自组装的最优化工艺条件的考察,为工业发酵优化表达人乳头瘤病毒病毒样颗粒提供了应用基础。更多还原

【Abstract】 The recombinant Pichia pastoris GS115/HPV16L1(muts)fermentation conditions for human papillomavirus the virus-like particles(HPV VLPs)were optimized.Based on the defined shaking flask medium,effects of pH of the culture medium,temperature,ammonium sulfate,and oleic acid were investigated during the induction period in human papillomavirus virus-like particles fermentation.The results showed that the monomeric protein L1 expression conditions were remarkably different from the conditions for assembly.During HPV L1 fermentation process,the optimized conditions for L1 expression are those:pH =4.5,temperature 28 ℃,and ammonium ion concentration 0.26-0.30 mol/L at the exponential growth phase.However,for VLPs production,the culture conditions are as follows:pH=3.5,temperature 30 ℃,and the ammonium ion concentration 0.34-0.42mol/L controlled at the exponentialphase.In addition,the monomer L1 expression was inhibited with the addition of oleic acid at the initial induction period.Oleic acid(φ=5%)could promote the monomer L1 self assembly at the middle induction phase.The relationship between the intracellular monomer L1 expression and its self assembly can be applied for the expression of the human papillomavirus virus-like particles by recombinant Pichia pastoris in industrial production.更多还原