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

目的：运用基因工程方法获得重组小鼠β-防御素2(mouse Beta Defensin 2, mBD2),体外实验观察mBD2对非分型流感嗜血杆菌( Non-typeable Haemophilus influenzae, NTHi)的抑菌活性,初步探讨mBD2的杀菌机制。方法①对工程菌Rosetta-gami(2)-pET32a(+)/mBD2进行诱导培养,采用亲和层析法对目的蛋白进行纯化,SDS-PAGE分析重组mBD2的分子量大小及表达情况。②在不同浓度的重组mBD2和不同浓度的NaCl条件下,不同作用时间内,观察重组mBD2对NTHi的体外抗菌活性；并用二硫苏糖醇处理重组mBD2,观察构象改变对重组mBD2抗菌活性的影响。③体外建立NTHi黏附A549细胞模型,镜下观察细菌黏附情况；菌落稀释培养法观察重组mBD2对NTHi黏附A549细胞的抑制；电子显微镜进一步观察重组mBD2引起的NTHi细胞损伤。结果①SDS-PAGE 分析表明,重组菌Rosetta-gami(2)-pET32a(+)/mBD2在分子量约4 kD处均有一条明显的表达带,与理论重组蛋白的分子量大小相符；经过酶切及纯化,每升工程菌培养物可获得约7.5 mg/L、具有较高纯度的mBD2成熟肽。②体外抗菌实验研究表明,纯化的重组mBD2体外具有明显的抗NTHi活性,培养120 min后,其最小抑菌浓度( MIC)值为40μg/ml,最小杀菌浓度( MBC)值为160μg/ml。外环境NaCl浓度的增高及重组蛋白二硫键的破坏会抑制其抗菌活性。③成功建立了体外NTHi黏附A549细胞模型,镜下观察到NTHi能明显黏附于A549细胞表面；40μg/ml的重组mBD2与NTHi、A549细胞共同孵育2 h,NTHi的细胞黏附率显著降低,下降至2.3%；电镜下观察到,与40μg/mL重组mBD2共同孵育120 min后,NTHi菌细胞的细胞膜变得不完整,出现孔隙,有少量内容物逸出。结论重组mBD2对NTHi具有杀菌作用,其杀菌活性除受本身的浓度和构象(二硫键)影响外,还受杀菌时间、外环境中无机盐浓度的影响,主要抗菌机制是通过损伤细菌细胞膜使细胞内容物外漏而最终杀灭细菌。

Purification of Recombinant mBD2 and Detection of Its Antibacterial Activity to NTHi

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.