制备大肠杆菌菌蜕的方法比较

Comparison of preparation methods for of bacterial ghosts from Escherichia coli

为进一步研究大肠杆菌菌蜕的制备技术,以pBV221、pCold IV和pETDuet1载体为基础构建了表达噬菌体E基因的溶菌质粒,并利用pETDuet1的2个多克隆位点构建了同时表达E基因和金黄色葡萄球菌核酸酶A的双表达溶菌质粒。经检测,pBV221-E/DH5α组和pCold-E/BL21组的裂解效率最高,但pCold-E/BL21组的裂解起始时间晚于其他几组。诱导剂的浓度对pETDuet1-E-SNA/BL21(DE3)的裂解效率有较大影响。电泳结果显示,随着E基因的表达,细菌DNA逸出膜外,同时金黄色葡萄球菌核酸酶A将DNA降解为250 bp以下的小片段。除pBV221-E/BL21组外,β-丙内酯可实现对其他试验组菌蜕的完全灭活。电镜观察发现,几组菌蜕在结构上并无明显差别。与对照菌相比,菌蜕结构完整,电子密度低且不均匀,细胞膜有不同程度的皱缩。

In order to further study the preparation technology of bacterial ghosts from Escherichia coli,the bacteriolytic plasmids expressing bacteriophage E gene were constructed based on vectors including pBV221,pCold IV and pETDuet1.Using two polychonal sites of pETDuet1,a double expression plasmid expressing E gene and Staphylococcus aureus nuclease A was constructed.The lysis efficiency of pBV221-E/DH5αgroup and pCold-E/BL21 group was the highest,but the lysis initiation time of pCold-E/BL21 group was later than other groups.The concentration of inducer had a great influence on the lysis efficiency of pETDuet1-E-SNA/BL21(DE3).Electrophoresis results showed that with the expression of E gene,bacterial DNA escaped from the cell membrane.Meanwhile,Staphylococcus aureus nuclease A degraded DNA to a small fragment below 250 bp.Theβ-propiolactone could completely inactivate the bacterial ghost of experimental groups expect pBV221-E/BL21 group.Electron microscopic observation indicated that there was no significant difference in the structure of the bacterial ghost among different groups.Compared with Escherichia coli,the electron density of bacterial ghost was lower and uneven,the cell membrane shrank,and the structure was complete.