内含肽介导的天蚕素AD基因工程表达、其酰胺化及应用  

Intein-mediated recombinant expression of Cecropins AD,its amidation and application

作　　者：周冠 李蕾 邓玮 李希平 俞超 

机构地区：[1]青岛康伦生物科技有限公司,山东青岛266000

出　　处：《中国病原生物学杂志》2016年第8期705-709,共5页Journal of Pathogen Biology

基　　金：科技型中小企业技术创新基金项目(No.14C26213712180)

摘　　要：目的设计、合成天蚕素AD(CAD)基因序列,与内含肽(Intein)融合表达后通过重组蛋白自剪切作用获得C端酰胺化的CAD,并与抗生素配伍使用。方法根据大肠埃希菌密码子偏好性对CAD基因序列进行改造,人工合成目的基因,构建重组质粒pCold-SUMO-CAD-Intein,转化大肠埃希菌,诱导表达重组蛋白,经镍柱亲和层析纯化,后用DTT及肠激酶切割,利用分子筛纯化获得酰胺化CAD,并进行电泳及质谱鉴定。采用微量肉汤法检测重组CAD抑菌活性及其与不同抗生素按不同比例配伍使用的抑菌活性。结果 PCR扩增目的基因片段为132bp,与预期一致。质谱显示表达的CAD分子质量单位为3.949ku,与理论值相符。所得CAD抗菌肽对常见革兰阴性菌抗菌力强,配伍抗生素使用能发挥协同抑菌作用。结论成功构建CAD基因重组质粒pCold-SUMO-CAD-Intein,通过与内含肽(Intein)融合表达,经分子内的自剪切作用直接获得C末端酰胺化的抗菌肽,与抗生素配伍使用,能显著降低抗生素用量,具有作为抗生素替代物的应用潜力。Objective The gene sequence of cecropin AD（CAD）was designed,synthesized,and expressed in E.coli by fusion with Intein in order to obtain C-terminal amidated CAD through self-splicing of the recombinant protein and to use CAD in combination with antibiotics. Methods The CAD gene sequence was modified using codons preferred by E.coli.The recombinant plasmid pCold-SUMO-CAD-Intein was constructed and expressed in E.coli.The expressed product was purified using Ni 2＋-affinity chromatography,treated with DTT,and cleaved with enterokinase.Gel filtration chromatography was used to obtain purified amidated CAD,and CAD was analyzed with electrophoresis and mass spectrometry.The antibacterial activity of CAD was evaluated in vitro using broth microdilution.The antibacterial activity of CAD was evaluated either alone or in combination with different antibiotics using the minimal inhibitory concentration（MIC）as a parameter. Results PCR amplification revealed that the target gene was 132 bp in length,which coincided with the expectations.SDS-PAGE results indicated that the fusion protein（molecular weight：77ku）was in a soluble form and that it accounted for 20% of bacterial proteins.Mass spectrometry results indicated that the molecular weight of purified CAD was 3.949 ku,which agreed with the theoretical value.Evaluation of antibacterial activity indicated that CAD had potent antibacterial action on common Gram-negative bacteria.The MIC of CAD 16ug/ml for E.coli CMCC44102,64ug/ml for O78,32ug/ml for O2,and 16ug/ml for O157.CAD displayed good synergistic effects when used in combination with different antibiotics. Conclusion The gene plasmid pCold-SUMO-CAD-Intein was successfully constructed and expressed in E.coli in a soluble form through fusion with Intein.C-terminal amidated CAD was obtained through direct protein self-splicing,and its antimicrobial activity matched that of commonly used antibiotics.Results indicated that the dosage of antibiotics can be significantly reduced when used in combination with CA

关 键 词：天蚕素AD 表达 酰胺化 抗生素 配伍 

分 类 号：R384[医药卫生—医学寄生虫学]