营养物脉冲高密度发酵工艺优化及其对猪圆环病毒2型Cap蛋白表达量的影响  

Optimization of pulsing-addition of high-density fermentation and its effect on the expression of Porcine circovirus type 2 cap protein

作　　者：李可乐 黄玉欣 刘鹏 田克恭 LI Kele;HUANG Yuxin;LIU Peng;TIAN Kegong(National Research Center for Veterinary Medicine,Luovang 471003,China;Publike Biological Engineering Inc,Luoyang 471003,China)

机构地区：[1]国家兽用药品工程技术研究中心,河南洛阳471003 [2]普莱柯生物工程股份有限公司,河南洛阳471003

出　　处：《黑龙江畜牧兽医》2021年第9期5-11,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：郑洛新自创区创新引领型产业集群专项“猪伪狂犬、圆环与肺炎支原体基因工程疫苗的研发与产业化”(201200211200)。

摘　　要：为了实现重组大肠杆菌的高密度发酵和猪圆环病毒2型Cap蛋白的高效表达,试验在10 L发酵罐中研究梯度恒速补料、恒溶氧补料、变速流加结合DO-stat补料这三种流加补料方式及营养物脉冲高密度发酵对质粒稳定性和目的蛋白表达量的影响,并选择搅拌转速作为主要控制参数进行500 L发酵罐工艺放大。结果表明:单纯采用流加补料的方式进行发酵,发酵过程中存在质粒丢失现象,质粒稳定性为50.3%~60.4%,影响目的蛋白的表达。在进行补料的同时,脉冲补加酵母提取物形成的营养物脉冲高密度发酵使质粒稳定性提高至95.0%以上。综合比较选择变速流加结合DO-stat补料下的营养物脉冲高密度发酵方案,实现了Cap蛋白的高效表达;10 L发酵罐菌体OD600值、菌体湿重和Cap蛋白表达量分别为103.70,164.60 g/L和1050 mg/L,按照单位体积发酵液所消耗的搅拌功率相等的原则实现了500 L发酵罐的工艺放大,500 L发酵罐菌体OD600值、菌体湿重和Cap蛋白表达量分别为100.90,160.70 g/L和1031 mg/L,与10 L发酵罐比较差异不明显。说明营养物脉冲高密度发酵可以提高发酵过程中的质粒稳定性,实现菌株的高密度发酵和目的蛋白的高效表达。To obtain high density fermentation of recombinant E.coli and high efficiency expression of the Porcine circovirus type 2 Cap protein,three feeding modes of gradient constant rate feeding strategy,constant dissolved oxygen feeding strategy,variable-speed feeding combined with DO-stat feeding strategy were studied in 10 L fermenter.The effects of pulsing-addition of high-density fermentation on plasmid stability and target protein expression were studied.The stirring speed was selected as the main control parameters for the scale-up of 500 L fermenter.The results showed that the expression of target protein was affected by the loss of plasmid in the fermentation process,which was carried out by simply speed feeding.The plasmid stability was 50.3%-60.4%.During the different feeding process,the plasmid stability was increased to more than 95.0%by pulsing-addition of high-density fermentation.By comprehensive comparison,the pulsing-addition of high-density fermentation strategy with variable-speed feeding combined with DO-stat fed-batch selected to realize the high efficiency expression of Cap protein.The OD600 value,wet weight and Cap protein yield of 10 L were 103.70,164.60 g/L and 1050 mg/L,respectively.The scale-up fermentation process of 500 L fermenter was successfully performed by constant stirring power per unit volume.The OD600 value,wet weight and Cap protein yield of 500 L fermenter were 100.90,160.70 g/L and 1031 mg/L,which were not significantly different from 10 L fermenter.It indicated that the pulsing-addition of high-density fermentation could improve the plasmid stability in the fermentation process and realize the high-density fermentation of the strain and the high-efficiency expression of the protein.

关 键 词：猪圆环病毒2型 CAP蛋白 质粒稳定性 营养物脉冲高密度发酵 工艺优化 

分 类 号：S852.65[农业科学—基础兽医学] Q591.2[农业科学—兽医学]