Huang Peng, Yang Zhifang, Bao Jianying, Zhang Ning, Li Wenshu
College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China. *Corresponding authors, E-mail:
College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Mar;33(3):320-325.
Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na, 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.
目的 通过色谱法从人精浆中纯化人鹅型溶菌酶2（HLysG2）并分析其酶学性质。方法 采用蛋白质免疫印迹分析精浆中HLysG2的分布。利用阳离子交换色谱、几丁质亲和色谱和尺寸排阻色谱对精浆进行目标蛋白分离。通过蛋白质免疫印迹分析和质谱（MS）鉴定纯化产物。用高效液相色谱（HPLC）分析纯度。然后，采用比浊法测定HLysG2的最佳pH、离子浓度和温度及其标准活性。通过菌落形成试验评估HLysG2的杀菌活性。结果 蛋白质免疫印迹分析证实精浆中存在HLysG2。通过三种色谱法从精浆中纯化出一种约21.5 kDa的蛋白质，经蛋白质免疫印迹分析和质谱鉴定为HLysG2。纯化产物的最终纯度高于99.0%，在pH 6.4、0.09 mol/L Na、30℃条件下，峰值酶活性达到13 800 U/mg。体外试验表明，HLysG2对溶壁微球菌、枯草芽孢杆菌和金黄色葡萄球菌有显著杀伤作用，但对铜绿假单胞菌和大肠杆菌无作用。结论 可通过色谱法从精浆中获得天然HLysG2。它对革兰氏阳性菌具有体外杀菌活性，提示其可能在男性生殖系统的固有免疫中发挥作用。
