C. difficile is the crucial reason for antibiotic-associated diarrhea. It is a class of gram-positive bacteria. C. difficile, isolated from those domestic and nondomestic animals, leads to diarrhea in cats, horses, dogs, and pigs. C. difficile toxin A (CDTA) and C. difficile toxin B (CDTB), two large exotoxins, are the main reason for their virulence. It is shown that systemic antibodies to CDTA and CDTB have effective protection against severe C. difficile infection. The two exotoxins include three different functional domains: central translocation (T) domain, N-terminal enzymatic domain, and C-terminal receptor binding domain (RBD). The T domain mediates import into host cells. The N-terminal enzymatic domain is made up of glucosyl-transferase (GT) and cysteine protease (CP) moieties. And the C-terminal RBD possesses 38 tandem repeats. Several types of research prove that recombinant CDT (RCDT) fragments lead to induce robust immunity against lethal challenges with C. difficile.
Fig.1 Structures of CDTA and CDTB. (CD BioGlyco)
CD BioGlyco has constructed the RCDTA with those repeating units at its COOH terminus. It recognizes the carbohydrate receptor of the host cells, elicits neutralizing Abs, and protects laboratory animals against challenges with both toxin A and C. difficile. We utilize RCDTA as a carrier protein for the conjugations of the Pneumococcal type 14 polysaccharide, the Shigella Flexneri type 2a polysaccharide, and the Escherichia Coli K1 type polysaccharide. We activate the pneumococcal type 14 polysaccharide, and the S. flexneri type 2a polysaccharide with cyanogen bromide and then derivatize them with adipic acid dihydrazide. Subsequently, they are combined with RCDTA by water-soluble carbodiimide condensation. E. coli K1 type polysaccharide is derivatized with adipic acid dihydrazide. Then the derivative is associated with succinylated RCDTA by treatment with 1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC). We detect protein with the folin phenol reagent and identify saccharides by anthrone and ninhydrin methods.
In addition, CD BioGlyco provides Polysaccharide Vaccine Development by conjugating the polysaccharide II (PSII), highly complex polysaccharides on C. difficile cell surface, to the two carrier proteins including RCDTA_B2 and RCDTB_GT fragments. We assess the occurrence of complete conjugation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We confirm the occurrence by the constitution of a broad smear and the accompanied extinction of the narrow band of the proteins. Then we depurate the glycoconjugates by size exclusion chromatography (SEC) to transfer unbound saccharides. High-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC-PAD) is useful for the analysis of protein content and free saccharide content.
Fig.2 Conjugation scheme of PSII-toxins. (CD BioGlyco)
CD BioGlyco possesses a strong Glyco™ Vaccine Development Platform to guarantee high-quality polysaccharide vaccine development. We have diverse carrier proteins for clients to develop conjugate vaccines. Our experts possess extensive experience in the field of glycoscience and are capable of tailoring the optimal solution based on your specific demands. Feel free to contact us if you would like to learn more about our services and receive detailed information.
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