NP-1融合蛋白在毕赤酵母中的中试发酵研究

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	NP-1融合蛋白在毕赤酵母中的中试发酵研究
Alternative Title	Study on pilot-scale fermentation of NP-1 fusion protein in Pichia pastoris
	黎凯科
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	NP-1融合蛋白毕赤酵母中试发酵条件优化菌体活力蛋白酶
Abstract	NP-1是实验室利用基因工程构建重组菌株表达的一种融合蛋白药物，能够抑制肿瘤新生血管生成，其由抗肿瘤药物小肽基因与HSA基因融合通过毕赤酵母表达。本论文将在摇瓶发酵实验基础上，进行NP-1融合蛋白在毕赤酵母中的中试发酵研究，为后续工业化生产提供技术支持。论文得到以下结果。重组毕赤酵母表达NP-1融合蛋白的中试发酵研究在10 L发酵罐中进行，OD600=18的种子液按10%接种量接入BSM培养基中，甘油分批发酵阶段和甘油补料发酵阶段培养条件为：温度30 ℃，pH 5.5，DO>20%。菌体湿重达到200 g/L开始向发酵罐中流加甲醇，诱导阶段发酵条件：温度26 ℃，pH 6.0，维持0.05 MPa罐压。采用恒速补加甲醇诱导策略诱导发酵72 h，NP-1融合蛋白表达量为320 mg/L。优化诱导发酵条件为：诱导阶段维持发酵罐罐压为0.056 MPa，当菌体湿重达到198.990 g/L时开始诱导表达，诱导过程中甲醇浓度维持0.874%，NP-1平均表达量达到450 mg/L，比优化前提高了40.6%。对发酵中试过程中菌体活力测定发现：甲醇诱导阶段菌体呼吸速率大大增加，诱导36 h菌体呼吸速率最大，是起始诱导时的2倍。种子接种时间点对诱导发酵阶段菌体呼吸速率有重要影响，最优接种时间点为48 h。甲醇会对酵母菌体产生一定毒害作用，加快菌体衰老，使用BMMY培养基发酵菌体能更快适应甲醇，死亡率降低。发酵上清液的Western-Blotting实验显示发酵过程中产生的小分子蛋白具有HSA免疫原性，另外，活性酶谱实验发现发酵过程中会产生多种可降解NP-1的蛋白酶，并会随着发酵积累。这些蛋白酶的存在将会严重影响目的蛋白的收率，因此，如何减少这些蛋白酶的产生或抑制它们的活性可能是今后的工作重点之一。
Other Abstract	NP-1 is a fusion protein drug expressed by a recombinant strain constructed by genetic engineering in the laboratory, which can inhibit tumor angiogenesis. It is expressed by Pichia pastoris by fusion of an antitumor drug small peptide gene and HSA gene. This thesis will conduct pilot-scale fermentation research of NP-1 fusion protein in Pichia pastoris based on the shake flask fermentation experiment, and provide technical support for subsequent industrial production. The paper got the following results. The pilot-scale fermentation study of recombinant Pichia pastoris expressing NP-1 fusion protein was carried out in a 10 L fermenter. The seed solution with OD600=18 was inserted into the BSM medium at a 10% inoculum amount. The glycerol fermentation stage and glycerol feed were added. The culture conditions in the fermentation stage are: temperature 30 ℃, pH 5.5, DO>20%. When the wet weight of the bacteria reached 200 g/L, methanol was added to the fermenter. The fermentation conditions of the induction phase were: temperature 26 ℃, pH 6.0, and tank pressure of 0.05 MPa. A constant rate of methanol supplementation was used to induce fermentation for 72 h, and the expression of NP-1 fusion protein was 320 mg/L. The optimized induction fermentation conditions are: maintain the fermenter pressure at 0.056 MPa during the induction phase, and start to induce expression when the wet weight of the bacteria reaches 198.990 g/L. During the induction process, the methanol concentration is maintained at 0.874%, and the average expression of NP-1 reaches 450 mg. /L, an increase of 40.6% compared to before optimization. Measurement of bacterial cell vitality during the fermentation pilot test found that the bacterial respiration rate was greatly increased during the methanol induction stage, and the maximum bacterial respiration rate was at 36 h after induction, which was twice that of the initial induction. The time of seed inoculation has an important effect on the respiration rate of the bacteria in the induced fermentation stage, and the optimal time of inoculation is 48 h. Methanol will have a certain toxic effect on the yeast cells and accelerate the senescence of the cells. Fermentation cells with BMMY medium can adapt to methanol faster and reduce the mortality rate. The Western-Blotting experiment of the fermentation supernatant showed that the small molecule protein produced during the fermentation process has HSA immunogenicity. In addition, the activity zymogram experiment found that a variety of proteases that can degrade NP-1 are produced during the fermentation process, and will follow Fermentation accumulation. The existence of these proteases will seriously affect the yield of the target protein. Therefore, how to reduce the production of these proteases or inhibit their activity may be one of the focus of future work.
Pages	67
URL	查看原文
Language	中文
Document Type	学位论文
Identifier	https://ir.lzu.edu.cn/handle/262010/460131
Collection	生命科学学院
Affiliation	生命科学学院
First Author Affilication	School of Life Sciences
Recommended Citation GB/T 7714	黎凯科. NP-1融合蛋白在毕赤酵母中的中试发酵研究[D]. 兰州. 兰州大学,2021.

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