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目的 :对已构建的人肽抗生素hPAB β 1～ 8拷贝串联基因工程菌进行筛选 ,并对其优势菌株进行发酵研究 ,为hPAB β的规模生产奠定基础。 方法 :对 1～ 8拷贝串联基因工程菌的目标蛋白表达率进行比较后 ,选择 2～ 5拷贝菌进行菌产量、目标蛋白表达率、包涵体溶解性、亲和层析效果比较 ,确定最佳多拷贝菌 ;对其摇瓶条件下发酵的培养液、温度、气体条件、IPTG诱导时机、浓度及时间进行研究。 结果 :在相同条件下 ,3拷贝菌产量(3.15 3g/L)最高 ,目标蛋白表达率 (2 7.8% )接近最高水平 ,包涵体能溶解完全 ;利用亲和层析能成功捕获融合蛋白 ,后者经羟胺裂解 ,可获得相对分子质量与合成hPAB β成熟肽大小相符的小肽 ,筛选出的优势菌传代稳定 ,其最佳摇瓶发酵条件为 37℃ ,16 0r/min条件下 ,在改良M 9 CAA培养液中培养至吸光值 (A60 0 )≈ 2 .5时 ,以终浓度为10 0 μmol/L的IPTG诱导表达 5h。 结论 :确定了 3拷贝菌为最佳多拷贝菌 ,并确定了其最佳摇瓶发酵参数。
OBJECTIVE: To screen a series of genetically engineered strains of hPAB β 1 ~ 8 copies of human antibiotic, and to study the fermentation of its dominant strains, which laid the foundation for the scale production of hPAB β. Methods: After comparing the target protein expression rates of 1 ~ 8 copies of tandem genetic engineering bacteria, 2 to 5 copies of bacteria were selected to compare the yield, target protein expression, inclusion body solubility, affinity chromatography results to determine the best Multi-culture bacteria were prepared. The culture medium, temperature, gas conditions, timing, concentration and time of IPTG induction in shake flask culture were studied. Results: Under the same conditions, the yield of 3-copy bacteria was the highest (3.15 3 g / L), the target protein expression rate (2 7.8%) was close to the highest level, and the inclusion body was completely dissolved. The fusion protein was successfully captured by affinity chromatography. After cleavage with hydroxylamine, the small peptides with the same molecular weight and the same size as the synthetic hPAB β mature peptide were obtained. The selected dominant bacteria were passaged steadily. The optimum shaking flask fermentation condition was 37 ℃. Under the condition of 160r / min, 9 CAA medium to absorb light (A60 0) ≈ 2.5, the expression was induced by IPTG at a final concentration of 10 μmol / L for 5 h. Conclusion: Three copies of bacteria were identified as the best multi-copy bacteria, and its optimum fermentation parameters were determined.