Expression of GST-3B fusion protein of Escherichia coli of Ee strain producing SLT-Ⅱe toxin and study on its biological activities and immunogenicity

Abstract:

Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3B. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund's incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5×OD₅₀ Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease.