【作者】 卢锦；

【导师】 孙启玲；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 血栓病是人类面临的一类主要疾病，包括急性心肌梗塞、脑血栓、肺静脉血栓、动脉血栓和缺血性休克等。其中急性心肌梗塞（AMI）的死亡率高达30％。溶纤治疗被认为是治疗血栓病最为有效的方法，寻找、研制效果好、副作用小的治疗药物一直是世界范围的热门课题。重组葡激酶（Recombinant staphylokinase，rSak）具有纤维蛋白特异性高、纤维蛋白原降解少、对血小板富集型血栓作用强以及价格便宜等优点，使其成为一种极具潜力的特异性溶栓剂。本研究rSak是以活性可溶性状态存在于细胞内，在前期研究的基础上，对E.coli生产rSak的发酵工艺及其下游纯化工艺进行了全面优化，建立了一套高效表达、分离纯化简便、回收率高的工艺，为工业化生产提供了可行性研究。其主要内容包括：Ⅰ．基因工程菌生产rSak的发酵工艺优化。Ⅱ．发酵产物的分离纯化、结构性质鉴定。Ⅲ．rSak原液活性分析。本研究的主要研究结果如下：Ⅰ、通过单因素摇瓶培养实验，研究了碳源、氮源、培养温度对菌体生长和rSak表达的影响，确定合适的摇瓶培养条件：葡萄糖为碳源，氮源为酵母粉和蛋白胨（1∶2），培养温度为30～32℃；Ⅱ、将摇瓶优化出的培养条件放大至发酵罐进行单批发酵，研究了诱导维持时间对菌体的生长和rSak表达的影响，确定最佳诱导维持时间应控制在3～4h，此时rSak表达水平为35％左右，蛋白表达量和菌体密度相对较高且杂蛋白较少；Ⅲ、采用正交法对大罐发酵培养条件进一步优化，研究发现四个因素对菌体密度和目的蛋白质的表达存在这不同程度的影响，其影响大小依次为：葡萄糖浓度＞饱和氧浓度＞氮源浓度＞pH，并进一步确定了最佳的发酵培养条件：葡萄糖浓度2％、氮源浓度1.5％、pH6.8和饱和氧浓度30％。采用此优化条件对基因工程菌进行连续三批发酵，蛋白质表达量和菌体密度都较高，平均菌体密度A₆₀₀为35.8，平均DCW为8.2g／L以及平均蛋白质表达量达到52％。Ⅳ、利用超滤系统进行粗纯化，大大缩短了生产周期，对制品起到提纯、浓缩和转换介质的作用；确立了最佳的纯化工艺：粗提制品经DEAE-Sepharose FF和SP-Sepharose FF两步柱层析获得重组葡激酶原液；将两种同一性质不同强度的阳离子交换柱进行对比，研究证明SP-Sepharose FF层析柱更适合于重组葡激酶的分离纯化；对SP-Sepharose FF柱层析洗脱条件进行优化，实验表明低盐浓度逐步梯度洗脱更易于去除杂蛋白、提纯目的蛋白质，蛋白质回收率达到32％，SDS-PAGE纯度高达99％；在柱层析时，上样速度和洗脱速度对蛋白回收率的影响较大，通过实验证明，DEAE-Sepharose FF层析时，上样速度以较快为宜，而SP-Sepharose FF层析时适合较慢的上样速度；在对SP-Sepharose FF层析柱洗脱时，洗脱速度宜较快。Ⅴ、经小试工艺放大，进行连续三批试生产并对rSak原液作进一步的检测。rSak经SDS-PAGE检测为单带，且与rSak标准品位置相同，通过SDS-PAGE和HPLC分析，其纯度均在99％以上，分子量为15.5kD，与文献报道的相符，分子内没有二硫键，分子为单链聚合多肽。三批原液紫外吸收光谱、抗生素残留和细菌内毒素检测结果均符合基因工程制品的规定要求。Ⅵ、采用人纤维蛋白溶解法测定rSak原液生物学活性，本方法灵敏较高，连续三批产品活性分别是2.2×10⁸、3.1×10⁵、2.8×10⁸IU／ml，活性均较高并且稳定，进一步证明了发酵和纯化工艺重现性好。更多还原

【Abstract】 Thrombus is the major leading life-threatening disease human faced, including acute myocardial infarction,pulmonic infarction,artery infarction,shock lacked of blood and so on. The mortality is more than 30% caused by acute myocardial infarction. Thrombolytic treatment is considered as the best way to cure thrombus,it is always a hot point to study better thrombolytic drugs with fewer side-effect in the world. It has been demonstrated that Recombinant staphylokinase （rSak） induces fibrinolysis specifically without fibrinogen depletion and has higher fibrinolytic activity compared with other plasminogen activators such as SK, urokinase and tPA. In addition,rSak has been shown to be more efficient than SK for the dissolution of platelet-enriched and retracted blood clots. Therefore in recent years,rSak has become a promising drug and stimulated much structural and protein engineering research.In this report,rSak was expressed in a soluble and active form. This study comprehensive optimize the fermentation of recombinant E.coli expressing rSak,and downstream process of rSak was investigated.Moreover,the results were helpful to be scaled up to the industrial production of rSak.The main contents include:I. Study on the optimizing of fermentation process.Il.Study on the purification and identification of rSak. III. Analyze the activity of rSak.The main results of our study include:I、The recombinant E.coli strain was incubated in the flasks and the optimum conditions for cell growth and expression of rSak were as follows:glucose,yeast extract:tryptone（1:2）,temperature 30-32℃.II、The given fermentation parameter was applied to Biostar 70 litre fermentation tank in a pilot scale. It was found that induction time affected cell growth and expression of rSak,on the same time,the optimum induction time was 3-4h.III、The pilot-scale fermentations were thoroughly studied by orthogonalexperiment design. During the fermentation process,the conditions of some expression factors,such as pH,DO saturation and supplement,et al,affected rSak expression. The results were as follows: glucose 2%,nitrogen resource 1.5%,pH6.8,DO saturation30%.Under these conditions,the average expression of rSak was 52%,A₆₀₀ was 35.8 and DCW was 8.2g/L.IV、Ultrafiltration was used to concentrate and desalt solutes retained by the membrane or to collect material passing through the membrane. Purification process was established starting with precipitation of 30%（NH₄）₂SO₄,Ultrafiltration, followed by ion-exchange chromatographies on DEAE-Sepharose FF and SP-Sepharose FF.Under these optimum conditions, protein yield was 32% and the purity analyzed by SDS-PAGE was over 99%.V、The production was repeatedly obtained with the stable expression, protein yield and purity of rSak.The purified rSak migrated as a single protein band on reduced/non-reduced SDS-PAGE was above 99%.Molecular weight of single peptide chain at reduced SDS-PAGE condition demonstrated 15.5 kD.By inspections of SDS-PAGE,HPLC,UV absorption spectrum,residual antibiotics and bacterial endotoxin,the sources of rSak from continuous three batch were up to the standard of biologics.VI、The activity of rSak was measured by using the fibrin plate method that was more rapid and sensitive than others. The respective results of activity were as follows:2.2×10⁸,3.1×10⁸,2.8×10⁸IU/ml.It was confirmed that the whole fermentation and purification process were suitable and stable during rSak production.更多还原