Antibodies against recombinant catalytic domain of lethal toxin of Clostridium sordellii neutralize lethal toxin toxicity in HeLa cells.
Lethal toxin of Clostridium sordellii (MLD 150 ng/kg) is one of the most potent Clostridial toxins and is responsible for most of the diseases including sudden death syndrome in cattle, sheep and toxic shock syndrome, necrotizing faciitis, neonatal omphalitis and gangrene in humans. Lethal toxin (TcsL) is a single chain protein of about 270 kDa. In the present study, 1.6 kb DNA fragment encoding for the catalytic domain of TcsL was PCR amplified, cloned in pQE30 UA vector and expressed in E. coli SG 13009. The expression of recombinant lethal toxin protein (rTcsL) was optimized and it was purified under native conditions using a single step Ni-NTA affinity chromatography. The purified recombinant protein was used for the production of polyclonal antibodies in mice and rabbit. The raised antibodies reacted specifically with the purified rTcsL and intact native lethal toxin on Western blot. The biological activity of the recombinant protein was tested in HeLa cells where it showed the cytotoxicity. Further, the polyclonal antibodies were used for in-vitro neutralization of purified rTcsL, acid precipitated C. sordellii and C. difficile native toxins in HeLa cells. Mice and rabbit anti-rTcsL sera effectively neutralized the cytotoxicity of rTcsL and C. sordellii native toxin but it did not neutralize the cytotoxicity of C. difficile toxin in HeLa cells.
Biotechnology Division, Defence Research & Development Establishment, Gwalior-474002, M.P, India.
Preetika
P
Arya
S
S
Ponmariappan
Lokendra
L
Singh
G B K S
GB
Prasad
D000906:Antibodies / Q000276:immunology / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology*; D020134:Catalytic Domain / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D047989:Paraclostridium sordellii / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans
D000906:Antibodies; D001427:Bacterial Toxins; C072292:lethal toxin LT, Clostridium sordellii