Characterisation of cloning and in vitro expression of enolase from Streptococcus suis 2
Abstract number: P917
Pan X.Z., Sun W., Wang C.J., Zheng F., Tang J.Q.
Objectives: To clone enolase (eno) gene and investigate some characteristic of its product Enolase (Eno) in Streptococcus suis serotype 2 (S. suis 2, SS2).
Methods: Using the Qiaexpress expression plasmids, the enolase gene was inducibly over expressed in E. coli to produce Enolase with a hexahistidyl N-terminus to permit its purification. Western-blot experiment and Enzyme-linked immunosorbent assay were used to detect its antigenicity. Whole cells ELISA was developed to give confirmation about Enolase localisation. Moreover, an adherence assay with Hep 2 cells was performed to determine the implication of the Enolase in the S. suis 2 adhesion. Action on lymphocytes designed to explore the role of Enolase in immunoreaction related to streptococcal infection.
Results: A highly homologous eno gene was unveiled by the genome-wide mining. The target DNA fragment of about 1.3 kb was successfully amplified using the genomic template of 05ZYH33 and protein expression analysis showed that a 75 kD protein can be observed in 12% SDS-PAGE, indicating that the recombinant 6His-fused Eno protein can be produced in E. coli under the induction of IPTG. Western-blot experiment demonstrated clearly it shares strong specific antigenicity. ELISA result suggested that Eno can occur on the surface of 05ZYH33 strain. Adherence assay showed a significant reduction in the adhesion of S. suis 2 in the 6His-fused Eno pre-incubated cells compared to the non-incubated cells. Evaluation of antibody titers in ELISA indicated that serum samples from pigs with SS2 infection have higher levels of antibodies that react with the recombinant Enolase than with whole bacteria sonication lysis and MRP. Apotosis of lymphocytes showed that streptococcal Enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.
Conclusion: The results exhibited the properties of Eno protein and provided an efficient Eno-based method for monitoring SS2 infection. Together, our data supported that Eno can function as a novel antigen, and may play pivotal roles in the severe infection of S. suis 2.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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