菌丝霉素在酪丁酸梭菌中的表达研究

Expression of Plectasin Gene in Clostridium butyricum

目的:构建菌丝霉素(Plectasin,Pt)的真核重组表达菌株,研究其在酪丁酸梭菌(Clostridium tyrobutyricum)中的表达和抑菌活性,提供一种高效,天然具有抑菌功能的新型食品添加剂和畜类养殖的抗菌制品。方法:先将启动子thl克隆到载体pMTL82151上,得到表达型质粒pMTL82151-thl,然后根据酪丁酸梭菌表达系统对密码子的偏爱性,人工合成优化后的Pt基因,再将产菌丝霉素基因Pt克隆到启动子thl的下游,构建表达质粒pMTL82151-Pt,并接合转化入酪丁酸梭菌(Clostridium tyrobutyricum),通过甲砜霉素抗性筛选阳性重组子,PCR鉴定,SDS-PAGE分析和琼脂孔穴扩散法筛选获得菌丝霉素组成型表达菌株,并进行工程菌酪丁酸梭菌(Clostridium tyrobutyricum)pMTL82151-Pt发酵表达研究。结果:thl基因与质粒pMTL82151连接,得到表达型质粒pMTL82151-thl,将产菌丝霉素基因Pt克隆到启动子thl的下游,成功构建表达质粒pMTL82151-Pt。实现了Pt基因在酪丁酸梭菌(Clostridium tyrobutyricum)中的组成型表达,SDS-PAGE分析显示,在4.4 kDa处有明显的目的条带。工程菌经发酵表达培养后取上清,对金黄色葡萄球菌ATCC25923的半抑制浓度(half-inhibitory concentration IC_(50))为12.74μg/mL。结论:本研究中成功将菌丝霉素在酪丁酸梭菌中表达,为以后大批量生产和推广菌丝霉素作为食品添加剂和肉食类畜牧养殖中抗生素替代品奠定了基础。

Objective:To construct a eukaryotic recombinant expression strain of mycelomycin(Plectasin,Pt)and study its expression level and antibacterial activity in Clostridium tyrobutyricum.A new type of food additive with high efficiency,green and antibacterial function will be provided.Method:The promoter thl was first cloned into the vector pMTL82151 to obtain the expression plasmid pMTL82151-thl,and then the optimized Pt gene was artificially synthesized according to the codon preference of the Clostridium butyricum expression system,and then the bacteria were produced.The silkworm gene Pt was cloned downstream of the promoter thl,and the expression plasmid pMTL82151-Pt was constructed and transformed into Clostridium tyrobutyricum.The positive recombinants were screened by thiamphenicol resistance,PCR identification,SDS-PAGE analysis and agar holes The constitutive expression strain of mycelium was obtained by diffusion method,and the Clostridium tyrobutyricum pMTL82151-Pt fermentation expression of the engineered strain Clostridium tyrobutyricum was studied.Result:The thl gene was ligated with the plasmid pMTL82151 to obtain the expression plasmid pMTL82151-thl.The mycelia gene Pt was cloned downstream of the promoter thl,and the expression plasmid pMTL82151-Pt was successfully constructed.Constitutive expression of the Pt gene in Clostridium tyrobutyricum was achieved.SDS-PAGE analysis showed a clear target band at 4.4 kDa.The supernatant of the engineered bacteria was expressed by fermentation and culture has a significant inhibitory effect on Staphylococcus aureus ATCC25923.The half-inhibitory concentration(IC 50)of Staphylococcus aureus ATCC25923 was 12.74μg/mL.Conclusion:The expression of mycelium in Clostridium butyricum was successfully applied in this study.It has laid the foundation for mass production and promotion of mycelium in the future as a food additive and an antibiotic substitute in meat animal husbandry.