重组人角质细胞生长因子-2工程菌的发酵条件研究  

Primary Studies on Fermentation Conditions of the Recombinant Human Keratinocyte Growth Factor-2

作　　者：杨诗明 梁波 王新星 冯红[1] Shiming Yang;Bo Liang;Xinxing Wang;Hong Feng(College of Life Sciences,Sichuan University,Chengdu Sichuan;Chengdu Farwits Biotechnology Co.,Ltd.,Chengdu Sichuan)

机构地区：[1]四川大学,生命科学学院,四川成都 [2]成都远睿生物技术有限公司,四川成都

出　　处：《生物过程》2018年第2期40-47,共8页Bioprocess

基　　金：成都远睿生物技术有限公司资助。

摘　　要：人角质细胞生长因子-2对烧伤、割伤等造成的皮肤损伤具有良好的促进修复和愈合功能,在临床、化妆品等方面具有极大的应用潜力。为此,人角质细胞生长因子-2的规模化生产和制备成为开发应用的一项重要前提。本文利用正交实验设计,在摇瓶条件下优化了培养基组分和pH等培养条件对大肠杆菌工程菌细胞生长和重组人角质细胞生长因子-2重组表达的影响。结果表明工程菌生长及表达的最优培养基组分和条件确定为葡萄糖10g/L、蛋白胨20g/L、酵母提取物10g/L、pH7.5及IPTG诱导时间5~6h。最后,在100升的BLBIO-15SIA发酵罐中以优化的发酵条件进行了3个独立批次的发酵实验,工程菌的生物量达到100g/L(DCW)、重组人角质细胞生长因子-2表达量占全细胞总蛋白约为30%。这些结果为进一步中试开发奠定了基础。The human keratinocyte growth factor-2 (hKGF-2) plays an important role in the proliferation of epithelial cells and wounding repairing, and then showing great application prospective in clinic therapeutics and cosmetic industry. Therefore, large-scale production and preparation of rhKGF-2 becomes a pre-requisite for the commercial exploitation. In this study, optimal fermentation conditions of the genetic engineering E. coli [Pet26b/KGF-2/BL(DE)21] that can recombinantly express the rhKGF-2, have been studied in the flasks by the orthogonal experiment design, including the medium components (glucose, tryptone, yeast extract and pH). The optimal conditions for the cell growth and rhKGF-2 expression were obtained as: glucose 10 g/L, tryptone 20 g/L, yeast extract 10 g/L, and induction time 5-6 h. Based on these data, fed-batch fermentation was carried out on the BLBIO-15SIA automatic fermenter. The dissolved oxygen concentration was maintained around 20%;and the recombinant cells were harvested at 4 h after IPTG induction. An average cell biomass was approximately 100 g/L (DCW) and the expression level of rhKGF-2 protein was achieved by about 30% of the whole-cell proteins. This result might be helpful for development of the pilot-scale fermentation of rhKGF-2.

关 键 词：人角质细胞生长因子-2 重组表达 发酵条件 正交实验设计 

分 类 号：Q7[生物学—分子生物学]