牡蛎抗菌肽Molluscidin的密码子优化、重组毕赤酵母表达及抑菌活性

Optimization and Recombinant Expression of Antimicrobial Peptide Molluscidin in Pichia pastoris and Its Antibacterial Activity

目的:利用重组毕赤酵母高效表达抑菌活性较好的牡蛎抗菌肽Molluscidin。方法:按照毕赤酵母密码子偏好性优化合成牡蛎抗菌肽Molluscidin,利用生物信息学方法分析其基本理化性质,经EcoRⅠ和NotⅠ双酶切后与表达载体pPICZαA连接,构建重组质粒pPICZαA-CgMoCo,电转至毕赤酵母X-33,利用博来霉素抗性和PCR筛选阳性转化子,利用甲醇诱导表达并结合SDS-PAGE和Western blot分析验证,通过甲醇浓度和培养时间优化诱导表达条件,利用滤纸片琼脂扩散法测定培养上清的抑菌活性。结果:获得优化核酸序列,生物信息学分析显示Molluscidin的预期分子量为6521.87 Da,等电点为11.28,在酵母体内半衰期>20 h,理化性质较为稳定;成功构建重组质粒pPICZαA-CgMoCo,目的基因成功嵌入毕赤酵母,SDS-PAGE和Western blot验证获得1株高效表达的重组酵母,其优化表达条件为30℃、250 r/min、1.0%甲醇诱导表达48~72 h,滤纸片琼脂扩散法初步证明重组Molluscidin对大肠杆菌、肺炎克雷伯菌等革兰氏阴性菌和金黄色葡萄球菌、枯草芽孢杆菌等革兰氏阳性菌均具有较好的抑菌活性。结论:筛选到1株重组毕赤酵母X-33/pPICZαA-CgMoCo,能高效表达抑菌活性较好的重组牡蛎抗菌肽Molluscidin,为其生产应用奠定了基础,也为重组贝类来源抗菌肽的开发利用提供了可资参考的技术途径。

Objective:To express antimicrobial peptide Molluscidin with high antibacterial activity in recombinant Pichia pastoris.Methods:The optimized nucleotide sequence of Molluscidin were synthesized according to P.pastoris codon usage frequency.The physicochemical properties were analyzed by bioinformatics.The fragment was ligated to pPICZαA vector after digested with EcoRⅠand NotⅠ.The recombinant expression vector was transformed into P.pastoris X-33 by electroporation.The recombinant strains were screened with Zeocin and identified by PCR.The recombinant Molluscidin was induced with methanol,and identified by SDS-PAGE and Western blot.The expression conditions were optimized by methanol concentration and culture time.The antibacterial activity was determined by disk diffusion test.Results:The optimized nucleotide sequence was obtained.Bioinformatics analysis showed that the predicted molecular weight was 6521.87 Da,the isoelectric point was 11.28,the estimated half-life in yeast was more than 20 h,and the peptide was classified as stable.The recombinant expression vector pPICZαA-CgMoCo was successfully constructed and transformed into P.pastoris.SDS-PAGE and Western blot demonstrated that one recombinant strain with high-level expression was obtained.The optimal expression conditions were 30℃,250 r/min,1.0%methanol for 48~72 h.Antimicrobial assay indicated that the culture medium supernatant containing recombinant Molluscidin had antibacterial activity against Gram-negative(i.e.,Escherchia coli and Klebsiella pneumoniae)and Gram-positive(i.e.,Staphylococcus aureus and Bacillus subtilis)bacteria.Conclusion:One recombinant P.pastoris X-33/pPICZαA-CgMoCo strain with high-level expression and antibacterial activity of recombinant Molluscidin is screened,which lays a foundation for its production and application,and provides a technical approach for the development of antimicrobial peptide from Mollusks.