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