Construction of recombinant eukaryotic expression plasmid harboring epsilon toxin gene of Clostridium perfringens and immunization studies for the development of DNA based vaccine

Abstract

The present study was aimed to develop a DNA based vaccine against the epsilon toxin of Clostridium perfringens which cause the fatal disease enterotoxemia in sheep. The findings of the study are summarized as follows: newlineand#61623; For the development of DNA based vaccine, PCR amplification of the gene encoding epsilon toxin was performed using gene specific primers. newlineand#61623; The epsilon toxin gene was cloned in pEGFPN-1 vector as GFP fusion and in vitro expression of epsilon toxin in CHO-K1 cells was confirmed by microscopic analysis. newlineand#61623; In another strategy for the expression of epsilon toxin in cytosolic and membrane bound form, Etx gene was PCR amplified. newlineand#61623; Epsilon toxin gene was cloned in pcDNA3.1 and pDisplay vectors for cytosolic and membrane bound expression of the protein. newlineand#61623; In vitro expression of epsilon toxin from DNA vaccine constructs (pcDNAEtx and pDSmEtx) in CHO-K1 cells was confirmed by immunoblotting using anti-Etx antisera. newlineand#61623; Mice immunized with pcDNAEtx (cytosolic) and pDSmEtx (membrane bound) DNA vaccine constructs mounted immune response against epsilon toxin. newlineand#61623; Epsilon toxin specific antibodies from the sera obtained after DNA vaccine immunized animals were able to detect epsilon toxin in western blot and ELISA. newlineand#61623; The Immunoglobulin isotyping analysis of the sera obtained by pcDNAEtx and pDSmEtx was performed and IgG1 isotype from heavy chain and Igand#1082; isotype from light chain was found to be dominant isotypes. newlineSummary newline76 newlineand#61623; No significant increase in titer was observed when mice immunized with pcDNAEtx, pDSmEtx were given 2 and 3 boosters newlineand#61623; To enhance the antibody titer different amount of immunization with higher concentration of pcDNAEtx and pDSmEtx did not show significance increase in titer. newlineand#61623; Immunization with pcDNAEtx or pDSmEtx DNA mixed with the adjuvant aluminum hydroxide did not increase the titer significantly. newlineand#61623; To analyze the efficacy of the sera obtained from DNA vaccine immunized animals to neutralize epsilon toxin in vitro, neutralization assay was performed and capacity of sera to protect MDCK ce