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目的从重组柞蚕抗菌肽AD毕赤酵母发酵液中分离纯化出抗菌肽纯品,并对其进行纯度、分子量和抗菌作用的鉴定。方法用离子交换和疏水层析法对发酵液中的重组柞蚕抗菌肽AD进行分离纯化,用高效液相色谱法分析产物的纯度,再用基质辅助激光解析—飞行时间质谱仪对产物进行分子量鉴定,并结合传统微量肉汤稀释法和流式细胞术分析重组柞蚕抗菌肽AD纯品对E.coli敏感株及耐药株的抗菌作用。结果重组柞蚕抗菌肽AD经离子交换和疏水层析纯化后的纯度达到98.40%,分子量为4068.15Da,与所设计的重组柞蚕抗菌肽AD大小相近。所纯化出的重组柞蚕抗菌肽AD对E.coli的敏感株ATCC25922和产β-内酰胺酶耐药株株ATCC35218的抗菌作用均为MIC 8μg/m L,而头孢他啶和氨苄西林对耐药株ATCC35218的MIC的值分别为8μg/m L和>32μg/m L。结论成功纯化分离出具有自主知识产权的重组柞蚕抗菌肽AD,其对E.coli敏感株和耐药株具有相同的抗菌效果。
OBJECTIVE To isolate and purify the pure antibacterial peptide from the recombinant Pichia pastoris AD Pichia pastoris fermentation broth and evaluate its purity, molecular weight and antibacterial activity. Methods The antibacterial peptide AD of recombinant tussah in fermentation broth was separated and purified by ion exchange and hydrophobic chromatography. The purity of the product was analyzed by high performance liquid chromatography. The molecular weight of the product was identified by matrix-assisted laser desorption-time of flight mass spectrometry The antibacterial activity of the recombinant Anthyma sp. Antimicrobial peptides AD against Escherichia coli-susceptible and drug-resistant strains was analyzed by traditional micro broth dilution and flow cytometry. Results The purity of recombinant Anthyma pestis AD was 98.40% after purified by ion-exchange and hydrophobic chromatography. Its molecular weight was 4068.15 Da, which was close to that of the designed recombinant Anthelmintic peptide AD. The antibacterial activity of the purified recombinant tussah antimicrobial peptide AD against E. coli strain ATCC25922 and the β-lactamase resistant strain ATCC35218 was MIC 8 μg / m L, while the antibacterial activity of ceftazidime and ampicillin against the resistant strain ATCC35218 MIC values were 8 μg / m L and> 32 μg / m L, respectively. Conclusion Recombinant Anthozoan peptide AD with independent intellectual property rights was successfully purified and has the same antibacterial effect against E. coli sensitive and resistant strains.