Biosynthesis of Siniperca chuatsi β-defensin based on recombinant Pichia pastoris and its antibacterial activity
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S917.1;Q786

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Grant from the National “13th Five-Year” Key Research and Development Program for Science and Technology Support (No. 2016YFD0400106)

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    Abstract:

    β-defensin are a series of cationic small molecule antibacterial peptides rich in cysteine. They play an important role in protecting mammals and aquatic organisms such as fish and shellfish from pathogenic microorganisms, and are important protein immune factors in the body's specific immune system. The primary structure of Mandarin fish (Siniperca chuatsi) β-defensin (ScBD) consists of a signal peptide region on the N-terminal and a mature peptide region on the C-terminal. The mature peptide is responsible for the biological activity of ScBD. The objective of the present study is to construct a recombinant Pichia pastoris strain producing Mandarin fish β-defensin was constructed to solve the source problem of small molecule antimicrobial peptide, a natural endogenous immune factor. The gene ScBD encoding Mandarin fish β-defensin mature peptide was isolated from the spleen of Mandarin fish by RT-PCR. After linking it with the expression vector pPICZαA, pPICZαA-ScBD was transferred to P. pastoris X-33. Positive transformants were screened by YPD plate containing high concentration of zeocin, and the expression was induced by 1% methanol for 96 h at 28℃, 250 r/min and pH 6.0. The products of recombinant P. pastoris were purified by nickel ion affinity chromatography,and the purified product was identified by MALDI-TOF/TOF mass spectrometry. The antibacterial activity of the recombinant strain products was detected by plate coating method and turbidimetric method. The results showed that the primary structure identification based on mass spectrometry demonstrated that the purified product was the expected recombinant ScBD with a molecular weight of 6.35 ku;The bacteriostatic test results showed that the inhibition rates of the recombinant strains products on Staphylococcus aureus, Bacillus subtilis and Vibrio parahemolyticus were 94.34%, 86.97% and 85.92%, respectively. The recombinant P. pastoris strain constructed could effectively synthesize the recombinant ScBD with biological activity, which provides a technical approach for further development of natural small molecule antimicrobial peptides from fish.

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吕星星,王莎莎,赵震,陶妍,谢晶,钱韻芳.基于重组毕赤酵母的鳜鱼β-防御素生物合成及其抑菌活性[J].上海海洋大学学报,2020,29(4):568-577.
LYU Xingxing, WANG Shasha, ZHAO Zhen, TAO Yan, XIE Jing, QIAN Yunfang. Biosynthesis of Siniperca chuatsi β-defensin based on recombinant Pichia pastoris and its antibacterial activity[J]. Journal of Shanghai Ocean University,2020,29(4):568-577.

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History
  • Received:October 17,2019
  • Revised:January 19,2020
  • Adopted:February 24,2020
  • Online: July 23,2020
  • Published:
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