重组人β防御素4的原核表达、纯化及抗铜绿假单胞菌活性初探

Prokaryotic expression and purification of human beta defensin 4 and its bactericidal activity against Pseudomonas aeruginosa

目的制备原核表达的重组人β防御素4(human beta defensin4,HBD4),初步探讨其对铜绿假单胞菌的体外抗菌活性。方法将编码HBD4的DNA片段连接到大肠杆菌表达载体pET42a中,构建好的表达质粒pET42a-HBD4转化到表达菌株BL21(DE3)中,0.5mmol/L IPTG诱导表达4h,亲和纯化融合蛋白后经Western blot鉴定,再以肠激酶酶切融合蛋白,镍柱亲和层析分离HBD4;采用打孔法初测其抗铜绿假单胞菌活性,并用微量稀释法测定HBD4与3种抗生素的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),比较其抗铜绿假单胞菌活性差异。结果GST-HBD4在大肠杆菌中以可溶形式成功表达,纯化的重组HBD4体外对铜绿假单胞菌有抗菌活性,其效能略低于亚胺培南,而高于哌拉西林和环丙沙星。结论采用基因工程技术成功表达了重组HBD4,其对铜绿假单胞菌有较强的杀菌活性,具有烧伤创面感染治疗的应用前景。

Objective To explore a new approach to express bioactive human beta defensin 4 （HBD4） in E. coll. Methods The gene fragment of HBD4 was artificially synthesized according to the sequence in Gen-Bank and then cloned into the pET42a vector. The recombinant pET42a-HBD4 was transformed into E. coli BL21 （ DE3 ） after verification by endonuclease restriction and sequencing analysis. The expression was induced by 0.5 mmol/L IPTG for 4 h, and the fusion protein was purified by Ni-resin chromatography and identified by Western blotting. The recombinant GST-HBD4 was digested by enterokinase and purified by Ni-resin chromatography again. Furthermore, the bactericidal activities of HBD4 against Pseudomonas aeruginosa were assayed in vitro. The minimal inhibitory concentrations （MIC） and the minimal bactericidal concentrations （MBC） of HBD4 against the bacterium were compared with Imipenem, Piperacillin or Ciprofloxacin. Results The GST- HBD4 fusion protein was expressed as a soluble protein in E. coli BL21 （DE3） successfully, and the purified HBD4 displayed antibacterial activity against Pseudomonas aeruginosa. Meanwhile, the antibacterial activities of HBD4 was superior to Piperacillin and Ciprofloxacin, but inferior to Imipenem. Conclusion The bioactive peptide of HBD4 is successfully expressed, and exhibits strong activity against Pseudomonas aeruginosa, which has the possibility for control of the burn would infection.