Functional and structural characterization of soluble recombinant epsilon toxin of Clostridium perfringens D, causative agent of enterotoxaemia

Abstract

Clostridium perfringens types B and D are responsible for enterotoxaemia, one of the major causes of cattle mortality and is therefore of great economic concern. The epsilon toxin produced by the organism is the major antigenic determinant and has been directly implicated for the disease causation. In the present paper, we evaluated the biological activity of the recombinant epsilon toxin (rEtx) produced as soluble protein in Escherichia coli. The rEtx was purified to near homogeneity by a one-step anion-exchange chromatography. The immunological identity of purified rEtx was confirmed by Western blotting using a monoclonal antibody against the native toxin. The rEtx formed heptamer in the Madin-Darby canine kidney (MDCK) cells and synaptosomal membrane of mouse brain and was cytotoxic to the MDCK cells with a CT(50) of 30 ng/ml. The rEtx was highly stable and its thermostability profile related well with its biological activity. The rEtx was purified in large amounts and exhibited all the properties of native toxin and therefore can be used for the development of vaccine against the pathogen.

Heterologous expression and biochemical characterization of recombinant alpha phosphoglucomutase from Mycobacterium tuberculosis H37Rv

“…For the expression of rPGM, the cells were induced with 0.25 mM isopropyl-b-D-thiogalacto-pyranoside (IPTG). The induced cells harvested at 8 h post-induction were processed as described by Mathur et al [7]. Soluble and insoluble fractions from the cell pellet were prepared as described earlier [7].…”

“…The induced cells harvested at 8 h post-induction were processed as described by Mathur et al [7]. Soluble and insoluble fractions from the cell pellet were prepared as described earlier [7]. The pellet was resuspended and solubilized in 10 mM Tris-HCl (pH 7.4) containing 8 M Urea.…”

Role of oxidative stress in facilitating carcinogenesis

“…[56][57][58][59][60] Alteration of ROS levels with the help of microbes suggested as a conserved strategy to tackle several diseases. Further, regulation of cellular ROS by microbes can be crucial in chronic inflammatory diseases such as cancer.…”

A low-toxic site-directed mutant ofClostridium perfringensε-toxin as a potential candidate vaccine against enterotoxemia

“…7,10,11 This method has been widely used in the investigation of recombinant vaccines. 7,[11][12][13] In this study, one or more essential amino acid residues of a target toxin are selected and substituted to decrease the toxicity.…”

“…16 MDCK cells are commonly used in cytotoxicity assay of ETX among the four sensitive cell lines to ETX, including MDCK, mpkCCDcl4 cells, and G-402, ACHN cells. 10,12,20,21 In this paper, our work focused on developing mutant-based ETX vaccines that could protect against ETX intoxication. We selected three amino acid residues (His106, Ser111 and Phe199) to construct mutant ETX (mETX) and selected the suitable one which has low or non-cytotoxicity and retained immunogenicity to be evaluated as candidate vaccine.…”