抗破伤风杆菌信息菌素Ph-CT的构建及其抗菌活性的初步研究

Bactericidal Activity of Constructed Recombinant Fusion Protein Pheromonicin-CT

目的构建和纯化抗破伤风杆菌信息菌素(Ph-CT),并初步检测其抗菌活性。方法从分泌针对破伤风杆菌表面抗原的单克隆抗体的杂交瘤细胞中扩增出抗体可变区基因序列,利用双联寡核苷酸点突变技术将抗体模拟物与大肠菌素Ia融合构建Ph-CT,经离子交换柱纯化后,通过菌落培养加入不同剂量信息菌素Ph-CT(终浓度2、4、8、16μg/mL),观察Ph-CT的体外抗菌活性。通过培养液中接种破伤风杆菌,并加入不同剂量的Ph-CT(终浓度4、16μg/mL),厌氧培养16h后取滤液及菌液行小鼠腹腔注射,观察Ph-CT的体内抗菌活性。结果成功构建抗破伤风杆菌Ph-CT。体外实验结果显示,涂布加入Ph-CT 2、4μg/mL孵育菌液的固体培养基上仍见破伤风菌落生长,而涂布加入Ph-CT 8、16μg/mL孵育菌液的培养基上均无菌落生长;体内实验结果显示,滤液和菌液组中对照组小鼠1d内均全部死亡,滤液组Ph-CT 4、16μg/mL组小鼠3d内全部存活,菌液组中Ph-CT 4μg/mL组小鼠3d内存活3只(50%),Ph-CT 16μg/mL组小鼠3d内全部存活。结论构建的Ph-CT在体内和体外都表现出对破伤风杆菌有抗菌活性,为临床治疗破伤风提供了新思路和新途径,具有应用于临床的潜在价值。

Objective To construct engineering peptide pheromonicin-Clostridium tetani（Ph-CT）,and to test its bactericidal activity.Methods We amplified the gene of variable regions from hybridoma cells which secreted monoclonal antibody（mAb）against antigen in the membrane of Clostridium tetani and linked the small antibody mimetic to the channel-forming domain of colicin Ia to create Ph-CT.The Ph-CT was purified by CM sepharose ion-exchange column.Its in vitro antibacterial activity was evaluated by colony culture with different doses of Ph-CT（final concentration 2,4,8,and 16μg/mL,respectively）.Then we inoculated culture medium with CT strains and different doses of Ph-CT（final concentration of 4and 16μg/mL）.The in vivo antibacterial activity of Ph-CT was evaluated by cumulative survival of mice.After 16hours＇ anaerobic culture,the mice was treated with filtered CT medium or CT medium.Results We constructed Ph-CT successfully.CT colonies appeared in the CT medium treated with Ph-CT（2,4μg/mL）,while no colony appeared in the CT medium treated with Ph-CT（8,16μg/mL）.All mice survived when they were injected with filtered CT medium treated with Ph-CT（4,16μg/mL）and CT medium treated with Ph-CT（16μg/mL）.Three（50%）mice survived when they were injected with CT medium treated with Ph-CT（4μg/mL）.All mice in the control groups died after CT infections.Conclusion PhCT may be of value as antibiotics against Clostridium tetani.