纳豆激酶检测体系的建立及其跨膜转运途径初探  

A Study on the Detection System and Transmembrane Transport Mechanism of Nattokinase

作　　者：李敏惠[1] 陈瑜[2] 蒋玉亮[2] 李艳[2] 余赟[3] 毛明星[2] 林定彪 杨亚琼[3] 杨平[3] 

机构地区：[1]成都医学院科研实验中心,成都610083 [2]成都医学院生物医学系,成都610083 [3]成都医学院基础医学院,成都610083

出　　处：《成都医学院学报》2016年第5期532-536,564,共6页Journal of Chengdu Medical College

基　　金：四川省教育厅项目(No:13ZB0220);四川省科技厅项目(No:2015JY0205);四川省卫生厅项目(No:130302;130298);成都医学院科研基金(No:CYZ11-005);国家级大学生创新项目(No:201313705007;201313705003;201413705005)

摘　　要：目的建立纳豆激酶（nattokinase,NK）酶联检测体系,探讨其跨膜转运机制。方法采用杂交瘤技术筛选抗NK的单克隆抗体,竞争抑制法鉴定单抗识别的抗原表位,Western Blot测定单抗特异性,双抗夹心ELISA法进行单抗灵敏度测定,Caco-2细胞模型探讨NK跨膜转运机制,生物信息学预测NK穿膜相关序列。结果筛选得到稳定分泌抗体且特异性识别NK的杂交瘤细胞6株,其中3株单抗识别相近的抗原表位,其余单抗识别不同的抗原表位;6株单抗检测NK的最小灵敏度分布为0.78~20.00ng/mL;Caco-2细胞模型显示,胞吞作用不是NK跨膜转运的主要途径;生物信息学提示,NK中含有3段穿膜相关序列。结论基于NK单抗建立的酶联检测体系,可有效监控NK跨膜转运过程,NK可能是依据蛋白转导结构域直接介导而实现其肠道吸收转运。Objective To establish the detection system of nattokinase（NK）and explore its transmembrane transport mechanism.Methods The anti-NK monoclonal antibodies were screened by hybridoma technology.The monoclonal antibody epitopes were identified by competitive inhibition assay.The monoclonal specificity was tested by Western Blot.The monoclonal antibody sensitivities were evaluated by the Sandwich ELISA method.The transmembrane segments of NK were revealed by bioinformatics.Results Six hybridoma cell lines with stable secrete and anti-NK antibodies were obtained,among which three monoclonal antibodies recognize similar antigen epitopes and the others recognize the different antigen epitopes.The minimum detection sensitivity of NK ranged from 0.78ng/mL to 20.00ng/mL.Caco-2 cell model results indicated that endocytosis was not the main way of NK transmembrane transport.Bioinformatics showed that NK contained three transmembrane segments.Conclusion The ELISA system for NK based on the monoclonal antibodies could monitor the transmembrane transport process of NK effectively and NK may cause its intestinal absorption with the mediation of the protein transduction domain.

关 键 词：纳豆激酶 单克隆抗体 跨膜转运 蛋白转导结构域 

分 类 号：R977.3[医药卫生—药品]