金黄色葡萄球菌噬菌体裂解酶Ply187的CHAP结构域的表达及抗菌活性分析  

Expression and Antibacterial Activity of CHAP Catalytic Domain of Staphylococcus aureus Phage Lysin Ply187

作　　者：吴蒙[1] 陆海荣[1,2] 黄青山[1,2] 

机构地区：[1]复旦大学遗传工程国家重点实验室,上海200438 [2]上海高科联合生物技术研发有限公司,上海201206

出　　处：《生物技术通报》2016年第9期232-238,共7页Biotechnology Bulletin

基　　金：中国博士后科学基金(2014M551322);上海市工业菌株工程技术研究中心(13DZ2252000)

摘　　要：噬菌体裂解酶是一种高效的抗菌蛋白,系统地分析噬菌体裂解酶催化域的活性,有助于设计高效杂合裂解酶。合成噬菌体裂解酶Ply187的CHAP催化结构域(CHAPPly187)基因,并构建表达载体p ET28a-CHAPPly187转化至大肠埃希菌BL21(DE3),经IPTG诱导表达和两步纯化,获得高纯度的CHAPPly187,再与溶葡萄球菌酶催化域(CATLysn)进行活性比较。比浊法检测显示CHAPPly187和CATLysn对金黄色葡萄球菌都具有较强的抗菌活性;CHAPPly187具有更宽的抗菌谱,最适作用p H范围更大,对离子强度的耐受能力更好,易受EDTA的影响。低浓度的二价金属离子Ca2+、Mg2+、Mn2+对两种催化域都有明显的促进作用,低浓度的Zn2+、Cu2+、Fe2+对它们有很强的抑制作用。Bacteriophage lysins are efficiently antibacterial proteins, thus systematically analysing the activity of catalytic domain of it is conducive to the design of efficient heterozygous lysins. The coding sequence of the CHAP catalytic domain of Ply187 （ CHAPptylS7 ） was synthesized, and a xecombinant expression plasmid pET28a-CHAPplylsTwas constructed, which then was transformed into Escherichia coli BL21 （ DE3 ） . The highly pure recombinant CHAPply187 protein was produced by IPTG induction, further purified by a two-step method, reaching 〉 95% purity, and finally compared with the catalytic domain of lysostaphin （ CATLyB. ） ~ The results from turbidimetry showed that both CHAPly187 and CATLyan possessed the solid antibacterial activities to Staphylococcus aureus. CHAPelyls7 showed a much broader antibacterial spectrum and optimal activity in a wider range of the pH, tolerated higher ionic strength, and more easily affected by EDTA. Ca2＋, Mg2＋, and Mn2＋ in low concentration significantly promoted the catalytic domains of both proteins, while the Zn2＋, Cu2＋, ahd Fe2＋ in low concentration strongly inhibited the catalytic domains.

关 键 词：噬菌体裂解酶 溶葡萄球菌酶 催化结构域 CHAP 抗菌活性 

分 类 号：Q55[生物学—生物化学]