Prokaryotic expression of Listeria monocytogene (LM) hly and development of monoclonal antibodies against listeriolysin O (LLO)

Abstract:

In order to study the pathogenesis of Listeria monocytogenes (LM), we cloned listeriolysin gene into prokaryotic expression vector PET21a. The expression vector was transformed into Escherichia coli BL21 for expression of listeriolysin O (LLO). LLO-His tag fusion protein was purified with a Ni-NTA affinity column and was used as an immunogen to vaccinate BALB/C mice. Hybridomas were developed by fusing mouse myeloma cells Sp2/0 and splenocytes from the immunized mice and screened with purified LLO. Three hybridomas secreting antibodies against listeriolysin O were obtained and named anti-LLO1, anti-LLO2 and anti-LLO3, respectively. Western blotting analysis showed that all of them could specifically bind to the LLO secreted by the LM. The titers of anti-LLO monoclonal antibodies in the supernatants of three hybridomas cultures were 1:3.6×104, 1:6.4×104 and 1:1.6×104, respectively, and the titers of ascites from the hybridoma-injected mice were 1:2×107, 1:2×107 and 1:1×107, respectively, based on ELISA test. The isotypes of the monoclonal antibodies were determined to be IgG1. The dissociation constants (Kd) of these three monoclonal antibodies were determined to be 6.18×10-11, 7.50×10-11 and 6.27×10-11 respectively. These data and reagents will be of great assistance to elucidate the pathogenesis of Listeriosis.