重组人BD3-BPI的内毒素中和活性及其在高盐环境中抗菌活性的稳定性

目的：验证重组人BD3-BPI（rhBD3-BPI）是否具有内毒素中和活性，研究其在高盐环境中是否能保持抗菌活性。方法：根据内毒素标准品绘制内毒素活性标准曲线，将100 μL梯度稀释的rhBD3-BPI-与100 μL 10EU／mL脂多糖（LPS）混匀，37℃水浴60min，同时设标准对照（只含10EU／mL LPS的标准品溶液），并以无热源水作为空白对照，采用基质显色法进行鲎试验测定LPS的活性；将6×10^8 CFU／mL的革兰阳性和阴性标准菌株及临床分离的多药耐药菌株接种于含1mg／mL rhBD3-BPI和0～250mmol／L不同浓度NaCl的液体细菌培养基中，37℃培养3h后用10mmol／L磷酸钠按1：1～1：1000的比例稀释，铺LB培养基平板，37℃过夜培养，观察各平板菌落生长情况，计数并计算杀菌率。结果：在5EU／mL的标准内毒素体系中，当rhBD3-BPI的浓度高于4μg／mL时即开始表现出一定的内毒素中和活性，当rhBD3-BPI的浓度分别为16、32 μg／mL时，其内毒素中和率分别为23％和88％，随后rhBD3-BPI对内毒素的中和活性趋于平稳，50 μg／mL的rhBD3-BPI对所有受检菌均表现出100％的杀伤效应。当NaCl浓度低于150mmol／L时，rhBD3-BPI对各受检菌的杀菌活性均未受明显影响；NaCI浓度升高至150-200mmol/L，rhBD3-BPI对各受检菌的杀菌活性有所下降，但其杀伤率仍在90％以上；当NaCl浓度高于200mmol／L时，盐浓度对rhBD3-BPI杀菌活性的影响才较为明显，但即使NaCl浓度达到250mmol／L，rhBD3-BPI的杀菌活性仍保持在85％以上。结论：rh-BD3-BPI具有内毒素中和活性，在高盐环境中具有良好的抗菌活性稳定性。

LPS Neutralization Activity and High-Salt Sensitivity of rhBD3-BPI

Objective： To study the lipopolysaacharide（LPS） neutralization activity and high-sah sensitivity of rhBD3-BPI, a recombinant human β-defensin 3（hBD3） and bactericidal permeability-increasing protein（BPI） fusion protein. Methods： Standard curve of LPS activity was created using Limulus Amebocyte Lasate test based on standard LPS product. 100 μL serially diluted rhBD3-BPI was mixed with 100 μL 10 EU/mL LPS and incubated in water for 60 minutes at 37℃. 10 EU/mL LPS served as control and water served as blank control. 6×10^8 CFU/mL standard or clinically isolated wild Gram-positive or-negative bacterial strains were incubated at 37℃ for 3 h in 1 mg/mL rhBD3-BPI supplemented with 0～250 mmol/L of NaC1 and then diluted at ratios from 1：1 to 1：1000 with 10 mmol/L sodium phosphate. 50 μL cells were plated in LB agar gel and incubated overnight, numbers of bacterial colonies were counted and bactericide rate was calculated. Results： In 5 EU/mL LPS system, LPS activity neutralization was detected when rhBD3-BPI was above 4 μg/mL, 23% and 88% LPS neutralization rates were reached when rhBD3-BPI was 16 and 32 μg/mL, respectively. 50 μg/mL rhBD3-BPI had a 100% bactericidal effect on all the strains. This effect was not influenced by increasing NaCl concentration before reaching 150 mmol/ L. When the concentration of NaCl reached 200 mmol/L, the bactericidal effect remained above 90%. Significant decrease only appeared when the concentration of NaCl was above 200 mmol/L, however, the bactericidal effect of rhBD3- BPI remained above 85% even at the NaCl concentration of 250 mmol/L. Conclusion： rhBD3-BPI can neutralize LPS and has a stable bactericidal activity in high-salt solutions.