兰州大学机构库 >生命科学学院
TMP融合蛋白在毕赤酵母中的中试发酵研究
Alternative TitleThe research of pilotscale ferment TMP fusion protein in Pichia pastoris
鲜军
Subtype硕士
Thesis Advisor李红玉 ; 李洋
2014-04-15
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline微生物学
Keyword毕赤酵母 HSA-TMP融合蛋白 大规模发酵 发酵优化 蛋白纯化
Abstract目前我国原发性血小板减少性紫癜(ITP)、继发性血小板减少性紫癜和化疗及骨髓移植等引起的血小板减少症正在逐年增加。传统治疗方法有输注血小板、激素治疗等,但这些治疗方法在临床中出现了免疫反应、二次传染等副作用,这给患者造成了身体伤害。TPO (促血小板生成素)是一种巨核细胞和血小板生成的主要调节因子。1994年,TPO分子被发现、克隆,并且开始进行临床开发,TPO基因药物由于在I、II期临床试验中发现给药后可产生与内源性TPO起交叉反应的中和性抗体,导致持久性血小板减少,在美国已停止进行临床试验。TMP (TPO模拟肽)是由14个氨基酸构成的TPO肽类模拟物,与TPO基因无序列同源性。研究表明TMP能够与TPO竞争性结合在c-mpl上有效促进血小板生成,TMP二聚体具有与促血小板生成素 (TPO)同样的功效,而且在人体内不会诱导产生与内源性TPO起交叉反应的中和性抗体。但是TMP存在半衰期短的缺点,无法在体内有效发挥促进血小板生成的功效。本论文以实验室构建的毕赤酵母 HSA-TMP 工程菌株为发酵菌株,在 20 L 发酵罐中对大规模表达 HSA-TMP 融合蛋白的发酵条件进行了研究。通过研究工程酵母生长曲线,基础盐发酵培养基对酵母生长的影响以及不同诱导条件对目的蛋白表达量的影响,得出了较佳的发酵条件:在发酵罐中,培养温度为 29 ℃,诱导温度为 27 ℃,pH=5 的条件下,通过对发酵罐各个参数的精细调节,在溶氧不低于 20 %的条件下,酵母菌在培养约 36 h 后(甘油耗尽 1 h 后)结束生物量积累阶段,进入甲醇诱导表达阶段;经过约 96 h 的发酵,菌体湿重可达到 500 g/L 左右,此时目的蛋白 HSA-TMP 的表达量为 1 g/L,且生物学活性最佳。本研究得到了利用毕赤酵母大规模发酵生产 HSA-TMP 融合蛋白的初步条件,为利用毕赤酵母表达系统生产第二代促血小板生成素提供了有价值的数据。
Other AbstractNowadays there have been thousands of people with thrombocytopenia caused by idiopathic thrombocytopenic purpura (ITP), secondary thrombocytopenic purpura and chemotherapy-induced thrombocytopenia in our country. Traditional therapeutic method such as platelet transfusion would cause side effects like immune antibody, secondary infection which damage to health of patients, and increase financial burden to families. Thrombopoietin (TPO) is a main regulatory factor in the production and differentiation of platelets and megakaryocytes. Thrombopoietin was cloned in 1994, then scientists gained much interests in its clinical applications. But because of neutralizing antibody produced in endogenous TPO cross reaction which cause reduce of platelet continuous, it has been stopped in clinical test in America. TMP is a TPO analogue with 14 amino-acids which have nothing nucleotide sequence homology to TPO. It is reported TMP can competitive bind to c-mpl and promote the production of platelet. TMP dimer have the same function as TPO, and also it would not induce neutralizing antibody. But short shelf life prevent its use in promoting platelet production. Fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics which conjugate the gene of polypeptides or protein with short shelf life to specific carrier protein gene. Fusion protein expressed by fusion gene still with functional properties also long shelf life. In present work, HSA-TMP fusion protein expressed in Pichia pastoris had been successfully constructed, and then a preliminary exploration for ferment condition of fusion protein expression in large-scale had been done. After the study of pichia growth curve, the effect of BSM in yeast grow, and different induction condition in the biomass of target protein. The optimal fermentation conditions: cultured at 29 ℃, induced at 27 ℃, pH=5, adjusting parameters to ensure DO ≥ 20 %. In first step pichia was cultured approximate 36 hours until glycerin exhausted, then into second step, methanol induced phase. After 96 hours fermentation, The biomass of pichia up to 500 g/L, and at this time the fusion protein HSA-TMP yield 1 g/L and the better bioactivity. Taken together, we obtain preliminary conditions for large-scale fermentation fusion protein HSA-TMP by Pichia expression system. we make a meaningful attempt for the thrombocytopenia in research and therapy.
Pages56
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/341941
Collection生命科学学院
Affiliation
生命科学学院
First Author AffilicationSchool of Life Sciences
Recommended Citation
GB/T 7714
鲜军. TMP融合蛋白在毕赤酵母中的中试发酵研究[D]. 兰州. 兰州大学,2014.
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