生物技术通报 ›› 2018, Vol. 34 ›› Issue (5): 163-171.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0823

• 研究报告 • 上一篇    下一篇

单增李斯特菌CdaA的抗原表位分析及抗体的制备

孙静娟1,邱景璇1,曾海娟1,丁承超1,王广彬2,李杰1,王淑娟1,刘箐1   

  1. 1. 上海理工大学医疗器械与食品学院,上海 200093;
    2. 徐州绿健乳品饮料有限公司,徐州 221006
  • 收稿日期:2017-09-29 出版日期:2018-05-26 发布日期:2018-06-07
  • 作者简介:孙静娟,女,硕士研究生,研究方向:单增李斯特菌的快速检测;E-mail:sunjingjuan9111@163.com
  • 基金资助:
    “科技创新行动计划”长三角科技联合攻关领域项目(15395810900),乳制品生产体系致病菌快速检测(3A15308006)

Antigenic Epitope Analysis and Preparation of Antibody of Listeria monocytogenes CdaA

SUN Jing-juan1, QIU Jing-xuan1, ZENG Hai-juan1, DING Cheng-chao1, WANG Guang-bin2, LI Jie1, WANG Shu-juan1, LIU Qing1   

  1. 1. School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093;
    2. Xuzhou Lüjian Dairy Co.,Ltd.,Xuzhou 221006;
  • Received:2017-09-29 Published:2018-05-26 Online:2018-06-07

摘要: 生物信息学分析结果显示单增李斯特菌腺苷酸环化酶CdaA具有良好的种内保守性、种间特异性和抗原表位结构,本研究对该蛋白的免疫原性进行验证并制备单克隆抗体,拟以CdaA为检测靶标检测单增李斯特菌。考虑到跨膜区可能会阻碍CdaA的表达,因而构建pET30a-Δ300cdaA并诱导表达Δ100CdaA。以纯化的Δ100CdaA制备的多克隆抗体效价可达1∶128 000。Western-blotting分析表明多抗能够识别从单增李斯特菌中提取的CdaA。另外,筛选得到一株单克隆抗体,3F8,单抗效价可达1∶512 000。Western blotting分析可知该单抗能够与9株单增李斯特菌和2株非致病李斯特菌的提取蛋白结合,并且与大肠杆菌、金黄色葡萄球菌、沙门氏菌等7株其他菌种的提取蛋白不结合,表明该单抗具有良好的特异性。利用生物信息学筛选检测靶标并分析抗原表位结构,最后成功制备多克隆抗体和单克隆抗体。

关键词: 单增李斯特菌, cdaA, 生物信息学, 抗原表位, 单克隆抗体

Abstract: Bioinformatics analysis showed that Listeria monocytogenes adenylate cyclase,CdaA,had solid intraspecific conservation,interspecific specificity,and antigen epitope structure. In this study,CdaA was used as a detection target to detect L. monocytogenes,and the immunogenicity of the protein was verified and monoclonal antibody was prepared. Given that the transmembrane domains might block CdaA expression,pET30a-Δ300cdaA was constructed and induced to express Δ100CdaA. The titer of polyclonal antibody prepared by the purified Δ100CdaA reached 1:128000. Western blotting analysis demonstrated that polyclonal antibodies recognized the CdaA extracted from L. monocytogenes. In addition,a monoclonal antibody,3F8,was screened,and its titer was 1∶512 000. Western blotting analysis showed that the 3F8 bound with the extracted proteins of 9 strains of L. Monocytogenes and 2 strains of non-pathogenic Listeria species,while not bound with the extracted proteins from 7 strains of other species,such as Escherichia coli,Staphylococcus aureus,and Salmonella,indicating it had promising specificity. In summary,we used bioinformatics methods to screen the detection target and to analyze the epitopes,and we prepared polyclonal antibody and monoclonal antibody successfully.

Key words: Listeria monocytogenes, cdaA, bioinformatics, epitope, monoclonal antibody