家蝇抗菌肽MAF-1A的串联表达与抗白念珠菌体外活性检测

Tandem expression and anti-Candida albicans activity determination of antimicrobial peptide MAF-1A from Musca domestica

目的构建家蝇抗菌肽MAF-1A基因原核串联表达体系,在大肠杆菌中表达具有抗菌活性的MAF-1A。方法根据大肠杆菌密码子的偏嗜性进行密码子优化,设计并合成含有5个拷贝的MAF-1A串联基因序列;将合成的串联基因序列克隆到表达载体pET28a并转化大肠杆菌Roseeta(DE3),应用IPTG诱导表达5×MAF-1A串联重组蛋白;通过SDS-PAGE电泳、Western Blot对表达产物进行分析;用肠激酶专一性切割经Ni-NTA纯化后的5×MAF-1A重组串联蛋白,得到MAF-1A单体;采用微量稀释法检测MAF-1A单体对白念珠菌的体外抗菌活性。结果 5×MAF-1A蛋白在大肠杆菌中呈可溶性表达,28℃、0.8mmol/L IPTG诱导12h可达最大表达量;酶切后的抗菌肽MAF-1A单体对白念珠菌的MIC和MBC分别为0.5mg/mL、1.0mg/mL。结论成功构建MAF-1A原核串联表达系统,重组表达的MAF-1A对白念珠菌具有较高的抑杀活性。

We constructed the prokaryotic expression system of antimicrobial peptide MAF-1A gene,expressing MAF-1A with antifungal activity in Escherichia coli.According to E.coli codon usage,we optimized cDNA sequence of MAF-1A.After that,the 5 multimeric MAF-1A genes were synthesized and then cloned into prokaryotic expression vector pET-28a.The combinant vector was transformed into E.coli Roseeta（DE3）and inducted with IPTG.The production was analyzed with SDSPAGE electrophoresis and Western Blot.The recombinant 5×MAF-1A protein was purified by use of Ni-NTA and cleaved specially by Enterokinase to generate the MAF-1A monomer.Antifungal activity of MAF-1A monomer against Candida albicans was determined with microdilution method.The 5×MAF-1A protein was successfully expressed in E.coli with solubility and the maximum expression was 2.0 mg/mL.The MIC and MBC of MAF-1Aagainst C.albicans were 0.5 mg/mL and 1.0 mg/mL respectively.The prokaryotic expression system of MAF-1A was constructed successful and could be expressed efficiently in E.coli cells with higher C.albicans activity.