Construct And Express Of Bovine Clostridium Perfringens Alpha-epsilon Fusion Toxin Gene And Its Immunologic Studies

Caused by Cl.perfringens type A in bovine disease occurs frequently,and Cl.perfringens type A is considered to be the main pathogen of bovine sudden death syndrome.In recent years, the reported of bovine enterotoxemia caused by Cl. perfringens type D moreand more common, because of the disease acute onset, short duration, high mortality, so,resulting ingreat economic losses to the livestock industry in china. alpha-toxin andepsilon-toxin as two major lethal toxins which are produced by Cl.perfringens types A and D.The research indicated that these two toxins are major immunogen of Cl.perfringens, and theycan stimulate the animals body to produce specific antibodies. This study was to clone andexpress alpha-toxin protein, epsilon-toxin protein and alpha-epsilon fusion protein in E. coli,and make sure this toxin proteins have biological activity.First of all, amplifed Cl.perfringens type A strain alpha-toxin gene and Cl.perfringens typeD strain epsilon-toxin gene respectively by PCR method, and then, the two genes were insertedinto plasmid pET-28a. At the same time, using the same restriction sites of the connectionmethod to construct a recombinant plasmid pET-28a, which containing alpha-epsilon fusiongene. All the constructed recombinant plasmids were transformed into BL21(DE3) PlysS hostbacteria after identified by double enzyme digestion and sequencing analysis and make sure theresults are all correct. After IPTG induction, the expressed products were detected bySDS-PAGE and Western blotting. Immobilized Ni-ion affinity chromatography was used topurify the target protein. Make the vaccine use the purify target protein then immunized mice.the serum antibody and cytokine secretion levels of mice were measured by ELISA. Finally, themice are all attacked by Cl.perfringens types A and D strains.As a result, this research has successfully constructed and expressed biologically activityalpha-toxin protein, epsilon-toxin protein and alpha-epsilon fusion protein. The result of ELISAexperiment detected significant antibody growth and decline curve after the mice wereimmunized by three proteins, and21days after the second immunization the growth anddecline curve reaching a peak. the secretion of IL-4and IFN-γ cell number compared with thecontrol group increased significantly (p<0.05). In Cl.perfringens type A challenged group, theimmune protection rate of alpha-toxin protein group, alpha-epsilon fusion proteins group and the control group were70%,80%and0%, Respectively; in Cl.perfringens type D challengedgroup, the immune protection rate of alpha-toxin protein group, epsilon-toxin protein group,alpha-epsilon fusion proteins group and the control group were60%,60%,90%and0%,Respectively.In summary, the study successfully expressed alpha-toxin protein, epsilon-toxin proteinand alpha-epsilon fusion protein in prokaryotic. Serum antibody levels were increased after usethree proteins immunized animals Respectively. Meanwhile, immunized animals can resist1MLD of Cl.perfringens types A and D strains attack. Fusion proteins demonstrated itssuperiority relative to the expression of individual proteins. The research will provide a newideas for the further development of genetically engineered multivalent subunit vaccine whichcan prevention cattle infected with different types of Cl.perfringens.