Expression, Purification Of Recombinant BoNT/ALC,BoNT/BHc2,BoNT/EHc2 Fragments And The Protective Effects Of RBoNT/EHc2

Botulinum neurotoxin (BoNT) consists of seven serotypes that have been assigned the letters A through G according to its antigenicity, and BoNT/A, B, E are responsible for most of human botulism. BoNT is divided into two parts structurally: Light chain(LC,50 kD) is the catalytic domain as zinc dependent metalloprotease; Heavy chain ( HC,100 kD ) is the binding and translocation domain. The carboxy-terminal portion of HC(Hc) plays a major role in interaction with the receptor located on target cells membrane and the Hc is divided into two subdomains additionally: Hcl and Hc2. At present, BoNT is one of the important toxins in biological warfare agents and of highly hazardous bioterrorism agents; therefore, it is of significance to perform an investigation into the recombinant BoNT antigenic fragments for developing diagnosis antibody and preventive vaccine.To express recombinant BoNT/A LC, BoNT/BHc2 and BoNT/EHc2, detoxic cultural Clostridium botulinum types A, B, E were used as templates to amplify BoNT/A LC, BoNT/BHc2 and BoNT/EHc2 gene fragments by PCR. Then the target genes were individually ligated to the pGEM-T vector and the sequences were determined. The identities of BoNT/A LC, BoNT/BHc2 and BoNT/EHc2 neucleotide sequences were above 99% compared with individual gene reported, and then they were digested by BamH I + Xho I or BamH I + Nhe I and ligated to prokaryotic vectors pET21a and pET28a digested with the same restriction endonucleases to construct corresponding recombinant expression plasmids pET21a-LC, pET28a-BHc2 and pET28a-EHc2. The resulting recombinant expression plasmids were respectively transformed into E.coli BL21(DE3)pLysS competent cells for expression induced by IPTG. Strong expression bands with relative mass of 53 000, 28 000, 20 000 were detected by SDS-PAGE, and the target proteins, namely the recombinant BoNT/A LC, BoNT/BHc2 or BoNT/EHc2 protein, accounted for 23%, 18%, 37% of total cellular proteins and mainly in inclusion bodies. The proteins rBoNT/A LC and rBoNT/EHc2 bearing 6xHis-tag were one-step purified to greater than 95% using chelating nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column. However, some smaller fragments were visible in SDS-PAGE gel of purified rBoNT/BHc2, and itshowed that this protein had an extend degradation in the process of purification. Western blot and indirect ELISA analysis revealed that the antiserum against native BoNT/A, BoNT/B, or BoNT/E had a specific affinity for the respective expressed recombinant protein and showed a good antigenicity.Among the purified three recombinant proteins, rBoNT/EHc2 was selected to evaluate its immunological protection for Kunming mice. After administrated the mice, the mice were not challenged with the cultural supernatant of C. botulinum types E until the antibody titer rose to a certain extent. The results showed the immunized mice can be completely protected when challenged by 2 MLD C. botulinum types E supernatant, and 50% (2/4) immunized mice protected when by 5 MLD, and all died when by 10 MLD and 20 MLD. It had statistically significant differences in survivals of immunized mice groups. Neutralization assay of mice serum against rBoNT/EHc2 presented similar results.