溶菌酶活性测定方法的改进及其在重组人溶菌酶质量标准建立中的应用  

Improvement of Lysozyme Activity Determination Method and Its Application in Recombinant Human Lysozyme Quality Standard Establishment

作　　者：宋纯艳[1,2] 张拓[3,4] 侯利平[3,4] 原剑[3,4] 黄亚娟[3,4] 韩治国[3,4] 刘曙晨[2] 甄蓓[2,3] 魏开华[2,3] 

机构地区：[1]中南大学药学院,湖南长沙410013 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]北京蛋白质组研究中心,蛋白质组学国家重点实验室,国家蛋白质科学中心(北京),北京102206 [4]北京正旦国际科技有限责任公司,北京102206

出　　处：《分析测试学报》2014年第8期917-921,927,共6页Journal of Instrumental Analysis

基　　金：国家高技术研究发展计划863项目(2012AA020205;2014AA020906);国家科技部重大仪器专项(2012YQ18011710);国家科技部传染病重大专项(2013ZX10002009-001-001)

摘　　要：获得一种趋于均相反应体系的比浊法,用于重组人溶菌酶活性的研究,并建立相关操作规程和质量标准.通过显微计数法进行底物菌液质控,改进酶与菌液的混合方式和最佳酶用量,采用“双直线法”优化反应计时区间,并精确测定重组人溶菌酶含量.显微镜菌体计数法确定0.3 mg/mL底物菌液中菌体数保持在1.1 ×108个/mL左右;酶与菌液在样品池内经吸排法快速混合可趋近均相体系;酶的最佳用量为3μg;确定反应计时区间为0～60s;依据所制定的酶活性操作规程,确定重组人溶菌酶活性质量标准为45 000～ 65 000U/mg,日内、日间相对标准偏差均小于5％;优选Lowry法并精确测定蛋白含量为（1.0±0.1）mg/mL.改进后的方法稳定性得到明显提高,且操作简便、结果可靠,已被3家研究单位、企业验证并接受.建立的重组人溶菌酶活性和含量测定质量标准及操作规程具有实际参考价值.A turbidimetric method which could verge on a homogeneous reaction system was obtained.The method was applied in study of recombinant human lysozyme activity,and the related operation procedures and quality standards were established.In this research,microscopic counting method was used for the quality control of substrate bacteria liquid.The mixing manner of enzyme and bacteria liquid was improved,so as the amount of enzyme used for detection.＂Double line method＂ was adopted to optimize the reaction time interval for activity detection.Also,the content of recombinant human lysozyme was detected accurately.By means of microscope count method,the bacteria number in reaction substrate was kept at about 1.1 × 108/mL,and the concentration of the reaction substrate was 0.3 mg/mL.To approach the homogeneous system,the reaction liquid of lysozyme and bacteria was mixed by sucking-expelling method in the sample pool.The optimum amount of lysozyme was 3 μg,the optimum reaction time was confirmed as 0-60 s.According to the operation procedure of enzymatic activity detection,the standard for activity of recombinant human lysozyme was confirmed as 45 000-65 000 U/mg,the RSD of intra-day and inter-day were both less than 5％.Lowry method was chosen for the content determination of recombinant human lysozyme,and the protein content was （1.0 ± 0.1） mg/mL.Stability of the optimized method was improved obviously,also,the operation was convenient and the detection result was reliable.Furthermore,the optimized method was verified and accepted by three research institutes and enterprises.The quality standards and operational procedures were established with important practical reference value.

关 键 词：重组人溶菌酶 比浊法 双直线法 Lowry法 

分 类 号：O657.3[理学—分析化学] O629.8[理学—化学]