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

Biosynthesis of Siniperca chuatsi β-defensin based on recombinant Pichia pastoris and its antibacterial activity

摘要:

鳜鱼β-防御素（Siniperca chuatsi β-defensin，ScBD）多肽链由N端的信号肽和C端的成熟肽组成，通过构建产鳜鱼β-防御素的毕赤酵母重组菌株，以解决天然免疫小分子抗菌肽的来源问题。通过RT-PCR从鳜鱼脾脏中分离编码β-防御素成熟肽的基因ScBD，将其与表达载体pPICZαA连接后构建重组表达载体pPICZαA-ScBD并转入毕赤酵母X-33；通过含高浓度博来霉素的YPD平板筛选阳性转化子后，于28℃、250 r/min和pH 6.0的条件下，使用1%甲醇诱导表达96 h；经镍离子亲和层析对重组毕赤酵母菌株的产物进行纯化，并对纯化产物进行MALDI-TOF/TOF质谱鉴定；通过平板涂布法和浊度法检测该重组菌株产物的抑菌活性。结果表明：基于质谱分析的一级结构鉴定证明纯化产物为分子量6.35 ku的预期重组ScBD；抑菌实验显示重组菌株产物对金黄色葡萄球菌（Staphylococcus aureus）、枯草芽孢杆菌（Bacillus subtilis）和副溶血性弧菌（Vibrio parahemolyticus）的抑制率分别为94.34%、86.97%和85.92%。本文构建的重组毕赤酵母菌株能有效合成具有生物学活性的重组ScBD，为鱼类来源天然小分子抗菌肽的进一步开发提供了技术途径。

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.