• EI
  • Scopus
  • 食品科学与工程领域高质量科技期刊分级目录第一方阵T1
  • DOAJ
  • EBSCO
  • 北大核心期刊
  • 中国核心学术期刊RCCSE
  • JST China
  • FSTA
  • 中国精品科技期刊
  • 中国农业核心期刊
  • CA
  • WJCI
  • 中国科技核心期刊CSTPCD
  • 中国生物医学SinoMed
中国精品科技期刊2020
马文君,郭校燕,滕琳,等. 重组人汗腺抗菌素蛋白的异源可溶表达、发酵优化及分离纯化研究[J]. 食品工业科技,2021,42(9):114−121. doi: 10.13386/j.issn1002-0306.2020080008.
引用本文: 马文君,郭校燕,滕琳,等. 重组人汗腺抗菌素蛋白的异源可溶表达、发酵优化及分离纯化研究[J]. 食品工业科技,2021,42(9):114−121. doi: 10.13386/j.issn1002-0306.2020080008.
MA Wenjun, GUO Xiaoyan, TENG Lin, et al. Soluble Prokaryotic Expression, Fermentation Optimization and Purification of Recombinant Human DCD-1L[J]. Science and Technology of Food Industry, 2021, 42(9): 114−121. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2020080008.
Citation: MA Wenjun, GUO Xiaoyan, TENG Lin, et al. Soluble Prokaryotic Expression, Fermentation Optimization and Purification of Recombinant Human DCD-1L[J]. Science and Technology of Food Industry, 2021, 42(9): 114−121. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2020080008.

重组人汗腺抗菌素蛋白的异源可溶表达、发酵优化及分离纯化研究

Soluble Prokaryotic Expression, Fermentation Optimization and Purification of Recombinant Human DCD-1L

  • 摘要: 目的:实现人汗腺抗菌素(Dermcidins,DCD-1L)的原核异源可溶表达,并进行纯化及活性分析。方法:首先检索NCBI确定人DCD-1L的序列,构建载体并转化E. coli ER2566,经异丙基-β-D-硫代半乳糖苷诱导表达后,进行SDS-PAGE电泳进行鉴定。其次,通过单因素实验,对不同温度、转速和补料速度等发酵条件进行研究与优化,根据DCD-1L蛋白特性优化蛋白纯化工艺。最后采用质谱和牛津杯法对纯化的蛋白进行分子量和抗菌活性鉴定。结果:经SDS-PAGE凝胶检测,在上清中分子量处检测到条带,证明采用原核表达系统可实现人DCD-1L的可溶表达;通过单因素实验获得最佳DCD-1L的发酵条件为:37 ℃培养,转速400 r/min,开启蠕动泵以50 mL/30 min补料速度自动加入发酵罐中;研究还建立了包括超滤、Q柱洗脱和分子筛在内的一整套优化的蛋白纯化工艺,获得的产品纯度大于96%,总回收率18.4%;最后,通过牛津杯法检测,纯化的重组人DCD-1L在浓度为50 μg/mL时,对表皮葡萄球菌这类革兰氏阳性菌较大肠杆菌具有更明显的抑菌活性。结论:本研究对重组人DCD-1L蛋白实现了原核可溶表达,并建立了稳定可靠的发酵、纯化工艺,并进行了初步的抗菌活性鉴定,为后续重组人DCD-1L蛋白的应用研究奠定了基础。

     

    Abstract: Objective: The study aimed to realize the prokaryotic soluble expression of human sweat gland antibiotic DCD-1L, optimize its fermentation and purification conditions, and analyze its antibacterial properties. Methods: Firstly, the sequence of human DCD-1L was identified by searching NCBI, and the vector pET28a-DCD-1L was constructed. After transformed into E. coli ER2566, it was induced by IPTG and identified by SDS-PAGE. Secondly, through single factor experiment, the fermentation conditions of different temperature, rotation and feeding speed were studied and optimized to determine the optimal fermentation conditions for DCD-1L. Thirdly, according to the properties of the protein, a complete purification process was established, including ultrafiltration, Q-column elution and molecular sieve. Results: The optimal fermentation conditions of DCD-1L were obtained: 37 ℃, 400 r/min, and the peristaltic pump was 50 mL/30 min. The purity of DCD-1L was more than 96%, and the total recovery rate was 18.4%. The purified recombinant human DCD-1L had more significant antibacterial activity against Gram-positive bacteria such as Staphylococcus epidermidis than Escherichia coli. Conclusion: The recombinant human DCD-1L protein is expressed in prokaryotic cells, and the fermentation and purification processes is stable and reliable, which lays a foundation for the industrial production of recombinant human DCD-1L protein.

     

/

返回文章
返回