抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究

denoregulin（ADR）是来源于南美树蛙Phyllomedusa bicolor皮肤的含有33个氨基酸的抗菌肽，在非极性环境中形成α_螺旋型结构，具有抗菌活性强、抗菌谱广的特点。将ADR基因克隆于pET32a载体上，转化大肠杆菌BL21(DE3)，对这一工程菌株的培养条件进行了优化。通过正交试验，考察诱导时机、诱导剂量和诱导时间三个因素的不同水平对蛋白表达的影响，结果发现诱导时机的影响尤为显著，考察了9种不同培养基对表达量的影响，发现培养基中加入葡萄糖对目标蛋白的稳定表达起了重要的作用，确定最佳培养条件为：培养基为2×YT+0.5%葡萄糖，诱导时机为OD600=0.9左右，诱导剂IPTG加入的终浓度为0.1mmol/L，诱导时间为4h。采用前期恒pH、后期指数流加的策略进行工程菌BL21(DE3)/pET32a-adr的高密度培养，在整个流加过程中，通过控制葡萄糖的加入量，将菌株的比生长速率控制在015h^-1，乙酸浓度也被控制在较低的水平(<2g/L)，但是质粒丢失严重，在发酵结束时，约有40%的大肠杆菌中不带质粒，这导致了目标蛋白的表达量下降严重，但是表达的目标蛋白90%以上为可溶性形式。表达的融合蛋白无抑菌活性，而裂解后得到的ADR单体具有明显的抑菌活性。

Optimization of Cultural Condition of Genetic Engineering Strain for Antibiotic Peptide Adenoregulin and Research on its Fed-batch Cultivation

33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.