解放军医药杂志Issue(2)：46-51,6.DOI:10.3969/j.issn.2095-140X.2016.02.012
重组小鼠β-防御素2的纯化及其对非分型流感嗜血杆菌抗菌活性的测定
Purification of Recombinant mBD2 and Detection of Its Antibacterial Activity to NTHi
姚锋 1张玉泉 1张建林 1杨益梅 1王姗姗 1陈程 1张俊荣1
作者信息
- 1. 226001 江苏 南通，南通大学附属医院妇产科
- 折叠
摘要
Abstract
Objective To obtain recombinant Beta Defensin 2 ( mBD2 ) of mouse using genetic engineering method and to detect its antibacterial activity to non-typeable haemophilus influenzae ( NTHi) in vitro in order to initially study mBD2 bactericidal mechanisms. Methods ①The Rosetta-gami(2)-pET32a( +)/mBD2 was cultured, and inter-est protein was purified using affinity chromatography. The molecular weight and expressions of recombinant mBD2 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE) . ②The antibacterial activities of recombinant mBD2 to NTHi in vitro were detected under different concentrations of recombinant mBD2 and NaCl within different culture times. The recombinant mBD2 was treated with Dithiothreitol, and the effect of conformational change on antibacterial activity of recombinant mBD2 was observed. ③A cell model of adherence NTHi to A549 in vitro was con-structed , and the bacterial adherence activity was observed under light microscope. The inhibition of adherence NTHi to A549 by recombinant mBD2 was observed using dilution plate method. The cell damage induced by recombinant mBD2 was further observed under electron microscopy. Results ①The SDS-PAGE analysis showed that the expression band of pET32a( +)/mBD2 in the Rosetta-gami(2) was obviously detected when the molecular weight was about 4 kD, which matched with theoretical value;every liter productivity of engineering bacteria culture reached 7. 5 mg/L after restriction enzyme and purification, and the obtained mBD2 mature peptide had high purity. ②The antibacterial test in vitro showed that the purified recombinant mBD2 had obviously antibacterial activity to NTHi. After 120 min of culture, the minimum inhibitory concentration ( MIC) of recombinant mBD2 to NTHi was 40μg/ml, and the minimum bactericidal concentration ( MBC) was 160 μg/ml. The increasing NaCl concentration and destroying DL-Dithiothreitol inhibited its antibacterial activity. ③The cell model of adherence of NTHi to A549 in vitro was established successfully, and NTHi was adhered to A549 obviously under light microscope observation. Adherence rate of NTHi to A549 was significantly dropped to 2. 3%after incubating in 40μg/ml recombinant mBD2 for 2 h. The membrane integrity of NTHi cell was affected, and pore ap-peared on membrane ,and some inclusion of cell escaped after incubating in 40 μg/ml recombinant mBD2 for 120 min. Conclusion Recombinant mBD2 has antibacterial activity on NTHi. The activity can be affected by concentration and configuration of recombinant mBD2, sterilizing time and concentration of inorganic salt. The main mechanisms of antibac-terial activity on NTHi of recombinant mBD2 make cytoplasm leakage by damaging cell membrane, and then the bacteri-um will be killed in the end.关键词
非分型流感嗜血杆菌/重组小鼠β-防御素2/抗菌活性Key words
Non-typeable haemophilus influenzae/Recombinant mBD2/Antibacterial activity分类
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姚锋,张玉泉,张建林,杨益梅,王姗姗,陈程,张俊荣..重组小鼠β-防御素2的纯化及其对非分型流感嗜血杆菌抗菌活性的测定[J].解放军医药杂志,2016,(2):46-51,6.
