人精浆中人鹅型溶菌酶2(HLysG2)的纯化及其酶学活性分析  

Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties

作　　者：黄鹏[1] 杨智昉[1] 鲍建瑛 张宁[1] 李文姝[2] 

机构地区：[1]上海健康医学院基础医学院,上海201318 [2]上海纽约大学文理学部,上海200122

出　　处：《细胞与分子免疫学杂志》2017年第3期320-325,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：上海市自然科学基金(15ZR1421800);上海市教育委员会科研创新项目(15ZZ116);上海市青年科技英才扬帆计划(15YF1408600)

摘　　要：目的从人精浆中分离纯化天然人鹅型溶菌酶2(HLysG2)并对其酶活性进行分析。方法通过Western blot法检测HLysG2在精液中的分布;分别使用阳离子交换层析、甲壳素亲和层析和分子筛层析技术从精浆中分离目的蛋白并利用Western blot法及质谱分析对其进行鉴定;使用高效液相色谱(HPLC)法分析纯化产物的纯度;使用比浊法测定HLysG2的最适pH值、最适离子浓度、最适温度和标准酶活性;使用克隆形成单位法测定HLysG2的杀菌活性。结果利用Western blot法在精浆中检测到HLysG2的存在;从人精浆中分离到相对分子质量(Mr)约21 500的目的蛋白并通过Western blot法及质谱分析鉴定其为HLysG2。纯化产物纯度达到99.0%以上。在pH6.4,30℃,Na+浓度为0.09 mol/L的条件下,HLysG2最高酶活性达到13 800 U/mg。体外检测显示HLysG2对溶壁微球菌、枯草芽孢杆菌和金黄色葡萄球菌具有杀菌作用,但对铜绿假单胞杆菌和大肠埃希菌的杀菌作用不显著。结论使用层析方法可以从人精浆中成功分离纯化到天然HLysG,其在体外具有对Gram阳性菌的杀菌活性,提示其可能在男性生殖系统的先天免疫中发挥作用。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 k Da 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＋,30℃. 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.

关 键 词：人鹅型溶菌酶2 精浆 纯化 杀菌活性 先天性免疫 

分 类 号：R392.33[医药卫生—免疫学] R378[医药卫生—基础医学]