重组毕赤酵母产青蛤Mytimacin抗菌肽的表达研究

Heterologous Expression of Cyclina sinensis Mytimacin Antibacterial Peptide Based on Recombinant Pichia pastoris

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。

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.