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食品与发酵工业  2021, Vol. 47 Issue (4): 1-6    DOI: 10.13995/j.cnki.11-1802/ts.025190
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响
钱晓芬, 吴涛, 赵理想, 孙杰, 汪钊, 魏春*
(浙江工业大学 生物工程学院,浙江 杭州,310014)
Effect of gene copy number on the expression of bovine lactoferrin functional fragment and cell survival in recombinant Pichia pastoris
QIAN Xiaofen, WU Tao, ZHAO Lixiang, SUN Jie, WANG Zhao, WEI Chun*
(College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China)
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摘要 为了提高牛乳铁蛋白功能片段(bovine lactoferrin functional fragment, BlfFf)产量,研究了基因拷贝数对重组毕赤酵母蛋白表达及酵母存活率的影响。将未折叠蛋白响应(unfolded protein response,UPR)激活因子基因HAC1、信号肽切割酶基因Kex2导入重组毕赤酵母BlfFfG01,并以核糖体rDNA非转录基因间隔区同源整合的方法结合PTVA(posttransformational vector amplification)法扩增重组菌株的基因拷贝数。利用SDS-PAGE、Western Blot、ELISA及流式细胞术分析多拷贝重组菌株发酵产物。分析表明,重组蛋白产量随着BlfFf基因拷贝数的增加而增加,但并非线性递增。HAC1拷贝数为3的BlfFfG12菌株产量相比单拷贝的BlfFfG10提高了150%,但更高的HAC1基因拷贝数降低了重组蛋白产量。流式细胞术分析显示,发酵末细胞存活率随着重组菌株中BlfFf基因拷贝数增加而下降,而HAC1基因拷贝数增加可一定程度上提高细胞存活率。摇瓶中BlfFf产量最高的BlfFfG12菌株(11拷贝BlfFf、3拷贝HAC1),在5 L罐进行高密度发酵,表达量为133.4 mg/L。BlfFfHAC1基因拷贝数对重组蛋白产量及细胞存活率影响显著,优化其基因拷贝数可有效增加重组蛋白产量。
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钱晓芬
吴涛
赵理想
孙杰
汪钊
魏春
关键词:  牛乳铁蛋白  毕赤酵母  HAC1  基因拷贝数  流式细胞术    
Abstract: The effects of gene copy number on protein expression in recombinant Pichia pastoris and yeast survival were investigated to increase the production of bovine lactoferrin functional fragments (BlfFf). The UPR activator HAC1 and signal peptidase Kex2 gene were introduced into recombinant P. pastoris BlfFfG01. Then, the gene copy number of the recombinant strains was amplified by homologous integration at the non-transcriptional gene spacer of ribosomal rDNA and PTVA (posttransformational vector amplification). SDS-PAGE, Western Blot, ELISA and flow cytometry were used to analyze the fermentation products by multi-copy recombinant strains. The results showed that recombinant protein production increased with BlfFf gene copy number, but the increase was nonlinear. The BlfFf production of BlfFfG12 strains with HAC1 copy number of 3 increased by 150% compared with that of single copy, but higher HAC1 gene copy number reduced recombinant protein production. Flow cytometry analysis showed, at the end of fermentation, cell survival decreased with the increase of BlfFf gene copy number in the recombinant strain. The increase of HAC1 gene copy number could improve the cell survival rate to some extent. BlfFfG12 strains with the highest BlfFf production in the shake flask culture (11 copies BlfFf, 3 copies HAC1), reached 133.4 mg/L BlfFf by high-cell-density fermentation in 5 L fermentor. In conclusion, the copy number of BlfFf and HAC1 genes had a significant effect on recombinant protein yield and cell survival rate. Optimizing the copy number could effectively increase recombinant protein yield.
Key words:  bovine lactoferrin    Pichia pastoris    HAC1    gene copy number    flow cytometry
收稿日期:  2020-07-28      修回日期:  2020-09-09           出版日期:  2021-02-25      发布日期:  2021-03-16      期的出版日期:  2021-02-25
基金资助: 浙江省自然科学基金(LY12B06010)
作者简介:  硕士研究生(魏春副教授为通讯作者,E-mail:chunwei@zjut.edu.cn)
引用本文:    
钱晓芬,吴涛,赵理想,等. 基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响[J]. 食品与发酵工业, 2021, 47(4): 1-6.
QIAN Xiaofen,WU Tao,ZHAO Lixiang,et al. Effect of gene copy number on the expression of bovine lactoferrin functional fragment and cell survival in recombinant Pichia pastoris[J]. Food and Fermentation Industries, 2021, 47(4): 1-6.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.025190  或          http://sf1970.cnif.cn/CN/Y2021/V47/I4/1
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