【作者】 迟文鹤；

【导师】 路福平；

【作者基本信息】 天津科技大学 ， 微生物与生化药学， 2005， 硕士

【摘要】 血栓栓塞性疾病严重威胁人类生命和健康,溶栓治疗是血栓性疾病安全有效的手段。目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵。因此研制高效、快速、副作用小,价廉的新型溶栓药物的需求迫切。微生物是获得溶栓剂的重要来源。 本论文在以前实验室小型发酵的基础上,对华根霉TK317(Rhizopus chinensis)的固体发酵工艺进行放大和优化,对发酵产物进行了粗提取,结果如下: 在1t固态发酵罐的发酵实验表明,产纤溶酶的最佳条件:初始湿度0.75L/kg含水量,发酵湿度70%,发酵前30h通风量640L/min,30h后通风量变为560L/min,28℃发酵时间60h,酶活力最高,达到706.3U/g物料。运用浸提、超滤浓缩、硫酸铵分级盐析、透析脱盐、冷冻干燥,对纤溶酶进行粗提取。纯化的样品经SDS-PAGE法验证。纤溶酶最终的比活力为134.84U/mg,纯化了27.91倍。 对编码华根霉纤溶酶的基因进行克隆,得到编码该纤溶酶的新基因,并将该基因在大肠杆菌pET表达系统中进行了表达,获得了重组蛋白。 根据已经测得的华根霉纤溶酶蛋白N端氨基酸序列,设计合理的简并性PCR引物。使用Trizol试剂提取华根霉的总RNA,经过RT-PCR,得到大小约为700bp的特异DNA条带,与pUCm-T载体相连,转化E.coli JM109,通过蓝白筛选、酶切鉴定、PCR验证得到转化子,测序,得到编码该酶的基因rcfe。与表达载体pET22b(+)连接,转化E.coli JM109,进行鉴定,提取转化子质粒,电转化E.coli BL21(DE3),通过PCR验证、诱导表达,对表达产物进行SDS-PAGE检验,证明华根霉纤溶酶基因获得表达。对重组蛋白进行酶活鉴定,证明重组蛋白没有纤溶酶活性。更多还原

【Abstract】 Thrombosis intimidates mankind’s life and health seriously, and fibrinolytic therapy is available. Current fibrinolytic enzymes available for clinical use are mostly plasminogen activators. Despite their widespread use, all these agents have undesired side effects and are very expensive. Therefore the search for novel fibrinolytic enzymes from various sources are being continued.The dissertation focuses on the breeding of Rhizopus chinensis producing a novel fibrinolytic enzyme. On the base of lab’s small-sized fermentation, solid fermentation technologies in the 1t biology reactor were optimized and rude extraction of fibrinolytic enzyme was carried out.The result on solid fermentation of Rhizopus chinensis indicated: the appropriate conditions were beginning moisture content 0.75L/kg substance, fermentation humidity 70%, initial ventilation 640L/min, 560L/min after 30h,28℃, fermentation cycle 60h, Productivity of the fibrinolytic enzyme went up peak value 706.3U/g by above conditions. The rude enzyme was purified with ultrafiltration concentration and ammonium sulfate fractionation. The specific activity was 134.84U/mg, 70.1% of the fibrinolytic enzyme was recovered with 27.91-fold purification. The rude enzyme was examined by SDS-PAGE.The expression of novel fibrinolytic enzyme in E.coli was investigated. According the N-terminal amino acid sequence of fibrinolytic enzyme protein, generate primers were designed. RNA of Rhizopus chinensis was extracted using TRIZOL reagent. The cDNA encoding fibrinolytic enzyme was cloned by RT-PCR, the cDNA was inserted into plasmid pUCm-T and transformed into E.coli JM109, the sequence rcfe encoding novel fibrinolytic enzyme was obtained. rcfe gene was cloned into plasmid pET22b(+) and transformed into E.coli BL21(DE3), resulting the vector pET22b(+)-rcfe. PCR and SDS-PAGE results indicated the fibrinolytic enzyme gene from Rhizopus chinensis has been successfully transformed and expressed in E.coli BL21(DE3). The recombinant protein was proved no fibrinolytic enzyme activity.更多还原