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目的在毕赤酵母中表达杂合抗菌肽CA(1-11)CD(12-37)ME(1-18)(ADM),并检测其抗菌活性。方法根据毕赤酵母(Pichia pastoris)偏爱密码子,合成杂合抗菌肽CA(1-11)CD(12-37)ME(1-18)基因,插入pPIC9K载体,构建重组表达质粒pPIC9K-ADM,电转化至毕赤酵母GS115,筛选阳性菌株,甲醇诱导表达,Tricine-SDS-PAGE检测ADM蛋白的表达,并对其抗菌活性进行初步检测。结果重组表达质粒pPIC9K-ADM经双酶切和测序鉴定证明构建正确;阳性酵母转化子的PCR产物可见目的基因条带;杂合抗菌肽在毕赤酵母GS115中获得分泌表达,可溶性蛋白约占菌体总蛋白的13%;且对E.coli DH5α、E.coliJM109和金黄葡萄球菌具有一定的抗菌活性。结论已在毕赤酵母GS115中表达了具有一定抗菌活性的杂合抗菌肽ADM,为相关新药的研究奠定了基础。
Objective To express the hybrid antibacterial peptide CA (1-11) CD (12-37) ME (1-18) (ADM) in Pichia pastoris and test its antibacterial activity. Methods According to the preference codon of Pichia pastoris, the cDNA of pCIC9K-ADM was constructed by inserting the gene of CA (1-11) CD (12-37) ME (1-18) into pPIC9K vector, The recombinant plasmids were transformed into Pichia pastoris GS115. The positive strains were screened and induced by methanol. The expression of ADM protein was detected by Tricine-SDS-PAGE and the antibacterial activity was also tested. Results The recombinant plasmid pPIC9K-ADM was confirmed by double enzyme digestion and sequencing. The PCR product of the positive yeast transformants showed the target gene band. The hybrid antibacterial peptide was secreted and expressed in Pichia pastoris GS115, 13% of the total body protein; and has certain antibacterial activity against E. coli DH5α, E. coli JM109 and Staphylococcus aureus. Conclusion The hybrid antibacterial peptide ADM with some antibacterial activity has been expressed in Pichia pastoris GS115, which lays the foundation for the research of related new drugs.