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本科毕业论文(设计)题目：重组大肠杆菌合成别藻蓝蛋白的发酵过程优化学院：医学院专业：生物技术姓名：赵耀飞指导教师：陈华新齐宏涛2012年5月25日-2-摘要嗜热藻别藻蓝蛋白是一种天然的荧光试剂，成熟三聚体三聚体具有较高的热稳定性且稀释后不发生解离。利用重组质粒构建表达载体，构建工程菌，使得嗜热藻别藻蓝蛋白在大肠杆菌中获得成功表达，而且目的蛋白的分离纯化效果较好。利用大肠杆菌大量表达别藻蓝蛋白需要的选择合适的培养基及发酵条件，应用中心组合设计进行实验设计，并运用响应曲面法进行数据处理，可以得到优化后的最适发酵条件。将此三种工程菌（分别表达别藻蓝蛋白的α亚基，β亚基，成熟三聚体）在装有15mL液体培养基的50mL摇瓶中的发酵过程进行优化。分析培养基起始pH值、培养温度、诱导起始时间和IPTG浓度对重组荧光蛋白表达量进行分析，可以得到多因子互作时的最优发酵条件。结果表明，单独发酵可以产生别藻蓝蛋白的α亚基和β亚基的菌种时，在pH值为7.2TB培养基，诱导温度为28℃，在发酵液OD值0.6加入IPTG（浓度为1mmoI／L），可以使亚基表达量达到45mg/L，在pH值为7.2TB培养基，诱导温度为28℃，在发酵液OD值0.6加入IPTG（浓度为1mmoI／L）三聚体表达量可以达到100mg/L。关键词：别藻蓝蛋白大肠杆菌发酵优化中心组合设计响应曲面法-3-AbstractAllophycocyanin(APC)，akindofnaturalfluorescentreagent,couldbeusedasfluorescentprobecombinedwithbiotinanddifferentantibodyinimmunodetections.Allophycocyanin(APC)trimerofThermosynechococcuselongtusBP-1isstableandindefectible.Escherichiacoliwithrestructuringexpressionvectorscouldproduceallophycocyaninandmakeiteasytobeextracted.Selectingtheappropriatefermentationconditionsisimportanttoeffectiveexpression.Theapplicationofcentralcompositedesign,andtheuseofresponsesurfacemethodologyfordataprocessing,theoptimizationoffermentationconditionscanbeoptimized.Fermentingthreekindofbacteriainflasks(50ml)whichcontain15mlliquidculturemedium.AfteranalyzingthedateofpHvalue,culturetemperature,inductionstarttime,andtheconcentrationofIPTG,wecangetthebestconditionthatcouldeffectivelyimprovethefermentationresults.Ifadd1mmol／LIPTGintofermentationliquidwhenitsopticsdensityis0.6andpHis7.2cultureitat28℃,volumeofproductionisabout100mg/L.Meanwhiletheproductionofαsubunitandβsubunitarebothmorethan45mg/Lunderthesamecondition..Keywords:AllophycocyaninEscherichiacolifermentationoptimizationcentralcompositedesign（CCD）responsesurfacemethodology（RSM）-4-目录1.前言......................................................................................................................................-6-1.1别藻蓝蛋白.....................................................................................................................-6-1.1.1别藻蓝蛋白简介......................................................................................................-6-1.1.2别藻蓝蛋白的结构与光谱性质................................................................................-6-1.2嗜热聚球藻别藻蓝蛋白在大肠杆菌中的生物合成与组装...............................................-8-1.3影响别藻蓝蛋白在大肠杆菌中表达的因素...................................................................-10-1.4响应曲面优化法与中心组合设计..................................................................................-11-1.5本实验研究的目的........................................................................................................-12-2.材料和方法.........................................................................................................................-14-2.1材料与试剂...................................................................................................................-14-2.1.1菌种.......................................................................................................................-14-2.1.2主要试剂...............................................................................................................-14-2.1.3培养基的配制........................................................................................................-14-2.1.4用于蛋白纯化的主要溶液......................................................................................-15-2.2抗生素及诱导剂...........................................................................................................-16-2.3主要仪器......................................................................................................................-16-2.4实验方法......................................................................................................................-17-2.4.1保种.......................................................................................................................-17-2.4.2发酵条件单因子预实验.........................................................................................-17-2.4.3中心组合设计优化发酵条件..................................................................................-19-2.4.4别藻蓝蛋白的分离纯化.........................................................................................-21-2.4.5蛋白浓度的转化....................................................................................................-22-3.实验结果与分析..................................................................................................................-23-3.1预实验实验结果...........................................................................................................-23-3.1.1培养基的选择实验结果.........................................................................................-23-3.1.2单因子限制变量预实验.........................................................................................-24-3.1.3荧光发射光谱峰值与蛋白浓度..............................................................................-26-3.2α亚基中心组合设计优化结果.....................................................................................-28-3.3β亚基优化结果....................................