基因工程白蛋白在毕赤酵母中的高效表达-生物化学与分子生物学专业论文.docxVIP

摘 摘 要 本文利用成功构建好的表达重组人血清白蛋白的Pichia pastoris菌株，对该表达系 统进行优化表达和纯化提取的研究，为基因工程白蛋白早日取代血液提取的白蛋白，满 足巨大的市场要求奠定基础。 在摇瓶培养规模研究表达株构建过程可能对蛋白表达量产生影响的因素： 载体整 合方式、整合拷贝数、甲醇利用表型。结果表明载体整合到酵母染色体的方式可以保证 表达单位的稳定遗传，菌株的甲醇慢速利用表型不影响蛋白表达量，由于能够在较高浓 度YPD+G418平板上生长的菌株，蛋白表达量较高，说明载体整合的拷贝数与表达量 正相关。故利用G418抗性筛选表达质粒拷贝数高的工程菌株，从而筛选出高效表达重 组人血清白蛋白的Pichiapastor菌株。 在5L NBS自动控制发酵罐中作发酵工艺学研究：用Plachett—Burman法结合因素 分析法进行高密度发酵工艺优化的研究：对两种甘油流加工艺作出比较；研究诱导表达 期甲醇流加速度控制方案。最佳培养基为无机盐基本培养基中加入0．1‰油酸，5 g／L 硫酸铵，l g／L柠檬酸，pH维持在5．5—5．9之间，甘油流加采用两步法即甘油流加与甘 油甲醇混合流加相结合。将筛选到的高表达株高密度发酵，细胞湿重达460 g几，发酵 液上清的人血清白蛋白含量达3．5 g／L以上。 对发酵表达产物进行纯化工艺研究，结合发酵培养基的特征，开发出一种流程简 单，易于放大，成本较低的纯化工艺：热处理，超滤，盐析，疏水层析和阴离子交换层 析。 ＼ ” 关键词重组人血清白1蛋白，Pichia Pastoris，高密度发酵，甘油流加工艺 发酵工艺优化，纯化工艺 AbstractTo Abstract To study the expression of recombinant human seium albumin in pichia pastoris and tO obtain high。level expression of rHSA with good biological activity． Numerous factors that influence heterologous protein production in a certain host system must be observed to gain successful gene expression and even with high yields，in pichia pastoris they includes：propenies of heterologous gene，site and method of intergration of the expression cassette，methanol utilization phenotype of the host gene dosage，stability of the product．0ur result showed that the secreting of rHSA in pichia pastoris was as high as O．599／L． Rapid optimization ofmicrobiological media by Plachett-Burman，a fuller analysis，together with some corresponding strategies and／or suggestion was done in 5L NBS Batch／continuous bioreactor culture．On the basis of the experiments the other factors such as optimal methanol feeding strategy，initial pH，optimal glycerol feeding strategy were also mentioned．In addition．the influences of the initial pH on protease activity were investigated．Using the improved medium．the concentration of rHSA in the fermentation 1iquid reached above 3．509／L． The supernatant was he-ated at 60℃for 8 hours and centrifugalized to get rid ofthe sediment．The heat—treated solution was tr