重组毕赤酵母产青蛤Mytimacin抗菌肽的表达研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0700

摘要/Abstract

摘要： Mytimacin是主要在无脊椎动物中表达的Macin抗菌肽家族中的一员,具有较强的抗病原微生物活性,是利用重组DNA技术开发天然抗菌剂的良好候选者。通过RT-PCR从青蛤（Cyclina sinensis）闭壳肌中克隆编码Mytimacin成熟肽的基因,经3次PCR在该基因的5'端添加Xho I限制性酶切位点和信号肽酶识别位点、3'端添加Xba I限制性酶切位点和6×His,获得目的基因“CsMm”以pPICZαA为表达载体、毕赤酵母（Pichia pastoris）X-33为工程菌,构建重组毕赤酵母X-33/pPICZαA-CsMm。通过高浓度博来霉素筛选高拷贝酵母转化子,在28℃、250 r/min条件下,使用1.5%的甲醇诱导表达72 h;使用固化金属离子亲和层析（IMAC）对表达产物进行纯化,并通过MALDI-TOF-TOF质谱分析对纯化产物进行鉴定。另外,通过涂布法和浊度法考察重组CsMm的抑菌活性。结果表明：基于X-33/pPICZαA-CsMm重组毕赤酵母的外源表达获得了表达量为25.6 mg/L的重组蛋白,经MALDI-TOF-TOF质谱鉴定其为分子量约7.8 kD的预期重组CsMm。抑菌试验证明重组CsMm对金黄色葡萄球菌（Staphylococcus aureus）、枯草芽孢杆菌（Bacillus subtilis）、大肠杆菌（Escherichia coli）和副溶血性弧菌（Vibrio Parahemolyticus）具有明显的抑菌活性。构建的重组毕赤酵母X-33/pPICZαA-CsMm能有效合成具有生物学活性的重组青蛤Mytimacin,旨为贝类来源天然小分子抗菌剂的开发提供可资参考的技术途径。

关键词: 青蛤, Mytimacin, 毕赤酵母, 重组表达, 抑菌活性

Abstract: Mytimacin is a member of the Macin antimicrobial peptide family expressed in invertebrates. It is a favorable candidate to develop natural antimicrobial agents by recombinant DNA technology because of its strong antimicrobial activity against pathogens. RT-PCR was used to clone the gene encoding mytimacin mature peptide from the adductor muscle of Cyclina sinensis;the Xho I restriction site and the signal peptidase recognition site were added to its 5' end,and the Xba I restriction site and a 6×His-tag to its 3' end after three times of PCRs the acquired target gene was named “CsMm”. The recombinant Pichia pastoris X-33/pPICZαA-CsMm was constructed using pPICZαA as an expression vector and P. pastoris X-33 as an engineering strain. The yeast transformants containing multicopy gene insertions screened by high-concentration zeocin were induced for 72 h with 1.5% methanol at 28℃ and 250 r/min,and the acquired culture medium supernatant was purified by immobilized metal affinity chromatography（IMAC）;the purified protein was identified by MALDI-TOF-TOF mass spectrometry analysis. In addition,the antibacterial activity of the recombinant CsMm was detected by coating method and turbidimetric method. The results showed that the heterologous expression based on the recombinant P. pastoris X-33/pPICZαA-CsMm a recombinant protein with an expression level of 25.6 mg/L;MALDI-TOF-TOF mass spectrometry analysis demonstrated that the purified recombinant protein was the expected recombinant CsMm with a molecular weight of 7.8 kD. The bacteriostasis test showed that the recombinant CsMm had obvious antibacterial activity against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Vibrio Parahemolyticus. The recombinant P. pastoris X-33/pPICZαA-CsMm constructed in the present study can effectively synthesize the bioactive recombinant C. sinensis Mytimacin,which provides a technical approach for the development of natural small molecule antibacterial agent from shellfish.

Key words: Cyclina sinensis, Mytimacin, Pichia pastoris, recombinant expression, antibacterial activity

赵震, 王莎莎, 吕星星, 陶妍, 谢晶, 钱韻芳. 重组毕赤酵母产青蛤Mytimacin抗菌肽的表达研究[J]. 生物技术通报, 2020, 36(5): 150-158.

ZHAO Zhen, WANG Sha-sha, LÜ Xing-xing, TAO Yan, XIE Jing, QIAN Yun-fang. Heterologous Expression of Cyclina sinensis Mytimacin Antibacterial Peptide Based on Recombinant Pichia pastoris[J]. Biotechnology Bulletin, 2020, 36(5): 150-158.

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