【摘要】 为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先采用易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造,将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库,以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb′042506。该蛋白使宿主菌体的湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序比对,该基因较原基因发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱显示该蛋白具有更强的特征吸收。更多还原

【Abstract】 To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5α. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.更多还原