【作者】 张怡洁；

【导师】 申烨华；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 本文包括五个章节。从发酵培养基,摇瓶培养条件,5L发酵罐培养条件三个方面对重组大肠杆菌DH5α生产rNGR-Tum-5的能力进行研究。使得在高密度培养情况下工程菌细胞终浓度OD₆₀₀达到16.0,rNGR-Tum-5表达量达到30%,rNGR-Tum-5的生产能力达到4.80。1.重组大肠杆菌DH5α发酵培养基的筛选在摇瓶中对大肠杆菌DH5α的发酵培养基进行研究,首先从几种常用的培养基中筛选出LB培养基作为基础培养基。在此基础上,对培养基中的碳源、氮源、微量元素进行筛选,得出在培养基组分（%）为:甘油1,酵母粉3,蛋白胨1,氯化钠0.5,微量元素母液0.15时,重组大肠杆菌DH5α生产rNGR-Tum-5的能力最强,较初始培养基提高了3倍。2.重组大肠杆菌DH5a摇瓶发酵的筛选在摇瓶中,研究发酵工艺条件如温度、溶氧量、种子菌龄、诱导时机、诱导剂浓度、诱导表达时间等对工程菌生长情况和蛋白表达的影响。得出最佳培养条件为:培养温度37℃,摇床转速200rpm,种子菌龄选择在OD₆₀₀为1.0,在对数生长中期(OD₆₀₀=2.0,4h处)时诱导,诱导剂浓度选择在0.5 mmol/L,诱导表达时间为4h。3.重组大肠杆菌DH5α在5L发酵罐中的培养根据工程菌高密度发酵的生长曲线,确定诱导时机。优选出合适的接种量8%,得出发酵罐中工程菌生长和蛋白表达情况。控制pH时采用二阶段培养法,前期菌体生长和后期蛋白表达维持在不同的pH范围;使工程菌生产rNGR-Tum-5的能力有所提高。对发酵产物进行分离纯化鉴定,在分子量约10⁴Da处出现一条蛋白带,为目标蛋白。更多还原

【Abstract】 The desertive consists of five chapters: The productivity of rNGR-Tum-5 from recombinant E.coli DH5αwas systematically studied, which includes optimization of fermentation culture medium, culture conditions in both shake flask and 5L fermenter, respectively. After high-density culture, the final cell growth of engineering bacteria OD₆₀₀ reached 16.0, the expression of rNGR-Tum-5 reached 30%, and the productivity of rNGR-Tum-5 from recombinant E.coli DH5αreached 4.80.1. Optimization of the culture media of recombinant E.coli DH5αTo improve the yield of rNGR-Tum-5 produced from recombinant E.coli, LB culture medium was chosen as the basic medium from several types of common medium. The influences of different types of carbon, nitrogen sources and trace elements in the culture medium on the yield of rNGR-Tum-5 had been investigated with shake flask method. It was found that the yield of rNGR-Tum-5 was the highest and it raised five times over that in the initial culture medium under the condition of 1% glycerol, 3% yeast extract, 1% peptone, 0.5% sodium chloride and 0.15% trace elements solution in the culture medium （w/w）.2. Culture of recombinant E.coli DH5a in shake flaskThe influences of fermentation conditions such as temperature, dissolved oxygen, inoculum age, induction time, inducer concentration, expression time on the growth of bacteria and protein expression has been studied. The best culture conditions were concluded as the following: culture temperature 37℃, shaking speed 200 rpm, inoculum age OD₆₀₀ 1.0, induction at mid-logarithmic growth (OD₆₀₀= 2.0, 4 h ), IPTG concentration chosen at 0.5mmol/ L, expression time for 4h. 3. 5L fermentor culture of recombinant E.coli DH5αInitially the appropriate seed amount of 8% was confirmed. The engineering bacterial growth and protein expression in fermentor were obtained. The ability of engineering bacterial producing rNGR-Tum-5 has been raised by controlling pH, which adoptted two-stage culture method. The pH in the early stage of cell growth was different from that in the late stage of protein expression, which raised significantly the yield of rNGR-Tum-5 . The product of fermentation has been separated and determined. The target protein appeared about 10⁴ Da in the molecular weight.更多还原