NP-1融合蛋白摇瓶发酵条件优化及种子库构建 | |

Alternative Title | NP-1 fusion protein shake flask fermentation conditions optimization and seed bank construction |

张涵姿 | |

Subtype | 硕士 |

Thesis Advisor | 李红玉 |

2021-05-29 | |

Degree Grantor | 兰州大学 |

Place of Conferral | 兰州 |

Degree Name | 理学硕士 |

Degree Discipline | 微生物学 |

Keyword | 毕赤酵母 融合蛋白 发酵条件优化 种子库 |

Abstract | HM-3是由内皮抑素衍生肽ES-2与小肽RGD通过linker相互连接而成的一段肽，具有明显的抗肿瘤作用。由本实验室构建完成的HM-3-HSA融合蛋白（NP-1）解决了HM-3在体内半衰期短的问题，但NP-1融合蛋白在摇瓶发酵中表达量仅有100-130 mg/L，距离NP-1融合蛋白商业化应用要求具有较大差距。因此，本文拟从培养条件出发探索NP-1融合蛋白摇瓶发酵的最佳条件，旨在提高蛋白表达量至300-500 mg/L，为发酵罐中试提供供试菌株，同时建立符合药典要求的酵母工程菌种子库。 实验探索了在甲醇浓度为2.0 %，诱导温度为30 ℃，蛋白诱导时间为120 h条件下，不同的菌体积累时间（12 h、24 h、36 h、48 h）对NP-1表达量的影响。结果显示在菌体积累时间为48 h时，NP-1表达量最高达到187.3 mg/L;实验还探索了在甲醇浓度为2.0 %，菌体积累时间为24 h，诱导温度为30 ℃条件下，不同蛋白诱导时间对NP-1表达量的影响。结果显示在诱导144 h时，NP-1表达量达到最高为195.0 mg/L。 实验探索了在菌体积累时间为24 h，诱导温度为30 ℃，蛋白诱导时间为120 h条件下，不同甲醇浓度（1.0 %、2.0 %、3.0 %、4.0 %）对NP-1表达量的影响。结果显示甲醇浓度为3.0 %时，NP-1表达量达到最高为200.6 mg/L;实验还探索了在甲醇浓度为2.0 %，菌体积累时间为24 h，蛋白诱导时间为120 h条件下，不同诱导温度（20 ℃、25 ℃、30 ℃、35 ℃）对NP-1表达量的影响。结果显示诱导温度为30 ℃时，NP-1表达量达到最高为188.7 mg/L。 实验探索了在甲醇浓度为2.0 %，菌体积累时间为24 h，诱导温度为30 ℃，蛋白诱导时间为120 h条件下，不同摇瓶装液量（10 %、15 %、20 %、25 %、30 %）对NP-1表达量的影响。结果显示装液量为20 %时，NP-1表达量最高为201.3 mg/L;同等条件下，实验也探索了不同摇瓶类型（P-P、P-D、D-P、D-D）对NP-1表达量的影响。结果显示摇瓶类型为D-D组合，即菌体积累和诱导阶段的摇瓶类型均为挡板摇瓶时，NP-1表达量最高为175.4 mg/L;同等条件下，实验也探索了不同转速（200 rpm、250 rpm、300 rpm）对NP-1表达量的影响。结果显示转速为300 rpm时，NP-1表达量达到最高为211.6 mg/L;同等条件下，实验还探索了透气性（M-M、M-S、S-M、S-S）对NP-1表达量的影响。结果显示透气性S-S组合，即菌体积累和诱导阶段摇瓶均用八层纱布封口时，NP-1表达量最高为175.5 mg/L。 在单因素实验基础上，设计了四水平三因素正交实验，综合甲醇浓度、菌体积累时间、诱导温度及蛋白诱导时间四个因素考察对NP-1表达量的影响。结果显示甲醇浓度为3.0 %，菌体积累时间为48 h，诱导温度为30 ℃，蛋白诱导时间为132 h时，NP-1表达量达到最高为304.1 mg/L。与未优化发酵条件前相比，NP-1表达量提高了168 %，并且表达量在其后的研究中一直稳定在300 mg/L以上。 本研究还建立了工程菌PichiaPink1#种子库，包括原始种子库、主种子库和工作种子库。种子库内各代次菌株在PAD平板上均呈现出典型酵母菌落形态;扫描电镜下酵母形态完整光滑，处于出芽状态酵母数量居多;通过PCR检测各代次菌株中NP-1基因，显示基因遗传稳定性较好;通过qPCR检测各代次菌株NP-1基因拷贝数，显示基因拷贝数均为1，未出现拷贝数下调情况;摇瓶表达结果显示种子库内NP-1的表达量均稳定在300 mg/L;以上结果均符合药典对种子库的鉴定要求。 关键词：毕赤酵母;融合蛋白;发酵条件优化;种子库 |

Other Abstract | HM-3 is a peptide composed of the endostatin-derived peptide ES-2 and the small peptide RGD connected to each other through a linker, which has obvious anti-tumor effects. The HM-3-HSA fusion protein (NP-1) constructed by our laboratory solves the problem of short half-life of HM-3 in vivo. However, when the NP-1 fusion protein was expressed in the Pichia pastoris system, the expression level of shake flask fermentation protein is only 100-130mg/L, which limits the commercial application of NP-1 fusion protein,there is a big gap between the requirements for commercial application of NP-1 fusion protein. Therefore, this article intends to explore the best conditions for NP-1 fusion protein shake flask fermentation based on the culture conditions, aiming to increase the protein expression to 300-500mg/L, provide test strains for the fermentation tank test, and establish compliance with the pharmacopoeia The required yeast engineering bacteria seed bank. The experiment explored the effect of different bacterial accumulation time (12h, 24h, 36h, 48h) on the expression of NP-1 under the conditions of methanol concentration of 2.0%, induction temperature of 30℃, and induction time of 120h. The results showed that when the bacteria accumulation time was 48h, the NP-1 expression reached the highest level of 187.3mg/L;The experiment also explored that when the methanol concentration is 2.0%, the bacteria accumulation time is 48h, and the induction temperature is 30℃. The effect of the induction time of NP-1 on the expression of NP-1. The results showed that NP-1 expression reached the highest level of 195.08mg/L at 144h of induction. The experiment explored the influence of different methanol concentrations (1.0%, 2.0%, 3.0%, 4.0%) on the expression of NP-1 under the conditions of 48h of accumulation time of bacteria, 30℃ of induction temperature, and 120h of induction time. The results showed that when the methanol concentration was 3.0%, the maximum expression level of NP-1 was 200.6mg/L. The experiment explored that at a methanol concentration of 2.0%, the bacteria accumulation time was 48 hours, and the induction time was 120 hours. Different induction temperatures (20℃, 25℃, 30℃, 35℃) on the expression of NP-1. The results showed that when the induction temperature was 30℃, the expression of NP-1 reached the highest level of 188.7 mg/L. The experiment explored the influence of different shake flasks (10%, 15%, 20%, 25%, 30%) on the expression of NP-1 under the conditions of a methanol concentration of 2.0%, a bacterial accumulation time of 48h, an induction temperature of 30℃, and an induction time of 120h. The results showed that when the liquid volume was 20%, the maximum expression of NP-1 reached 201.3mg/L;Under the same conditions, the effects of different shake flask types (P-P, P-D, D-P, D-D) on the expression of NP-1 also were explored. The results showed that when the shake flask type was a combination of D-D, that is, the strain accumulation and induction stage shake flask type is baffled shake flask, the highest expression level of NP-1 is 175.4mg/L;Under the same conditions, the effects of different speeds (200rpm, 200rpm, 250rpm, 300rpm) on the expression of NP-1 also were explored. The results showed that the maximum expression of NP-1 was 211.6mg/L when the speed was 300 rpm;Under the same conditions, the effects of air permeability (M-M, M-S, S-M, S-S) on the expression of NP-1 also was explored. The results showed that when the air-permeable S-S, that is, the bacteria accumulation and induction phases were sealed with eight layers of gauze, the highest expression of NP-1 was 175.5mg/L. Based on the single-factor experiment, a four-level three-factor orthogonal experiment was designed, combining the four factors of methanol concentration, bacterial accumulation time, induction temperature and induction time to investigate the influence on the expression of NP-1. The results showed that when the methanol concentration was 3.0%, the bacteria accumulation time was 48h, the induction temperature was 30℃, and the induction time was 132h, the maximum expression of NP-1 was 304.1mg/L. Compared with the unoptimized fermentation conditions, the expression of NP-1 was increased by 168%, and the expression level has been stable above 300mg/L in subsequent studies. This research also established the seed bank of engineered bacteria PichiaPink1#, including the original seed bank, the main seed bank and the working seed bank. All generations of strains in the seed bank showed typical yeast colony morphology on PAD plates;the morphology of yeasts was intact and smooth under scanning electron microscope, and most yeasts were in budding state;PCR was used to detect NP-1 gene in each generation of strains, showing genetic stability Good;the copy number of NP-1 gene of each generation of strains was detected by qPCR, showing that the gene copy number was all 1, and there was no copy number down-regulation;the expression results of shake flask showed that the expression level of NP-1 in the seed bank was stable at 300mg/L;The above results meet the requirements of seed bank identification. Key words: Pichia pastoris;fusion protein;optimization of fermentation conditions;seed bank. |

Pages | 80 |

URL | 查看原文 |

Language | 中文 |

Document Type | 学位论文 |

Identifier | https://ir.lzu.edu.cn/handle/262010/459929 |

Collection | 生命科学学院 |

Affiliation | 生命科学学院 |

First Author Affilication | School of Life Sciences |

Recommended CitationGB/T 7714 | 张涵姿. NP-1融合蛋白摇瓶发酵条件优化及种子库构建[D]. 兰州. 兰州大学,2021. |

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