流感疫苗神经氨酸酶的分离纯化方法建立及其参考品制备

Isolation and purification of neuraminidase from influenza vaccine and reference substance preparation

目的建立流感疫苗神经氨酸酶分离纯化方法,并制备神经氨酸酶定量检测参考品。方法制备对神经氨酸酶具有特异亲和力的功能化磁粒,建立基于该磁粒分离纯化神经氨酸酶的方法,优化分离纯化条件,对分离纯化的神经氨酸酶进行纯度分析和特异性验证,并进行酶活性测定和蛋白质定量。结果成功制备4-氨基苯草氨酸修饰的功能磁粒,建立基于该磁粒分离纯化神经氨酸酶方法,分离纯化神经氨酸酶纯度为98.7%,从H1N1、H3N2、B/Victoria和B/Yamagate 4种疫苗单价原液分离纯化的神经氨酸酶蛋白质浓度分别为71.50、100.58、64.11和37.68μg/ml,且均保留酶活性。结论建立了分离纯化流感病毒神经氨酸酶方法并制备了相应参考品。

Objective To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods Functional magnetic particles with specific affinity for neuraminidase were prepared.The method for separation and purification of neuraminidase was established based on the magnetic particles.The separation and purification conditions were optimized.The purity of neuraminidase was analyzed and the specificity was verified.The enzyme activity was determined and the protein was quantified.Results The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established.The purity of neuraminidase was 98.7%.The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1,H3N2,B/Victoria and B/Yamagate vaccines were 71.50,100.58,64.11 and 37.68μg/ml,respectively,and the enzyme activity remained.Conclusions The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.