重组柞蚕抗菌肽AD的纯化及鉴定

Purification and identification of recombinant Cecropin AD

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 separate and purify Cecropin AD from fermentation broth and to identify its purity, molecularweight and antibacterial action. Methods The ion exchange method and thin layer chromatography were adopted to purifycecropin AD from Pichia pastoris fermentation broth. The purity of purified products were analyzed by HPLC. Then themolecular weight of Cecropin AD was measured by MALDI- TOF/MS. The broth microdilution method and flow cytometry（FCM） were used to identify the antibacterial action of purified Cecropin AD. Results The purity of Cecropin AD thatpurified from Pichia pastoris fermentation broth reached 98.40%. The molecular weight was 4068.15 Da and approached thedesigned size. MIC of Cecropin AD to ATCC25922 of E.coli strain sensitive to beta-lactamase and ATCC35218 of E.coli strainto beta- lactamase were both 8μg/ml, while MIC of CAZ and AMP to ATCC35218 were 8μg/ml and more than 32μg/ml.Conclusion Cecropin AD with the independent intellectual property rights was separated and purified successfully. It hasthe same antibacterial action sensitive quality control stand and resistant E.coli strain.