人伤寒沙门菌Ty21a菌蜕的制备

Preparation and identification of Salmonella enterica Ty21a bacterial ghosts

目的探讨利用噬菌体PhiX174 E基因制备人伤寒沙门菌Ty21a菌蜕的可行性以及适当制备条件。方法将PhiX174 E基因克隆至温控表达系统,筛选获得裂解效率较高的质粒pMBE,通过优化转化方法将pMBE导入Ty21a,利用温控表达系统进行Ty21a菌蜕制备。菌落计数计算菌蜕裂解效率,并通过扫描电镜和透射电镜观察其形态结构。结果裂解质粒pMBE介导大肠杆菌DH5α的裂解效率达99.99%;通过电击转化方法获得重组Ty21a(pMBE),利用温控表达系统成功制备Ty21a菌蜕,裂解效率达96.77%。电镜观察菌蜕结构完整并呈空泡状结构,并能看到溶菌孔道。结论 PhiX174 E基因表达可实现人伤寒沙门菌Ty21a裂解形成Ty21a菌蜕,为其作为疫苗和药物递送载体的研究奠定了基础。

In order to prepare Ty21a bacterial ghosts accomplished by the temperature-controlled expression of bacteriophage PhiX174 lysis gene E, two recombinant lysis plasmids were constructed by cloning the lysis gene E into temperature-induced plasmid system, and their lysis efficiency to E. coli DH5a was compared by counting the colony forming unit （CFU）. Plasmid pMBE was transformed into bacteria Ty21a using improved transformation condition, then Ty21a bacterial ghosts （Ty21a BGs） were produced by shifting the temperature from 28℃ to 42℃. Ty21a BGs were harvested by centrifugation, and the lysis rate was calculated. The characterization of BG was observed by scanning electron micrograph （SEM） and transmission electron micrograph （TEM）. The lysis rate of E. coli ghosts which were induced by plasmid pMBE was about 99.99%. Bacteria Ty21a was efficiently transformed using optimal electroporation parameters （voltage 500V, electric resistance 2000, and capacitance 25μF）, forming recombinant Ty21a （pMBE）. Ty21a BGs were produced by controlled expression of PhiX174 lysis gene E, and the lysis rate was 96. 770/00. Ghosts show loss of cytoplasmic material and structural integrity. The Ty21a bacterial ghost was successfully produced by the controlled expression of PhiX174 lysis gene E, which provides an efficient delivery system for vaccine development.