Preparation and characterization of antigenic properties of gramicidin A- keyhole limpet hemocyanin and gramicidin A- ovalbumin conjugates

A rapid, simple and low cost procedure for preparing hapten-protein conjugates was developed using gramicidin A (GA) and two other water-soluble proteins, keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). GA was a kind of antimicrobial peptides. Two lysines and a cysteine were linked to amino- terminus and carboxyl-terminus of the peptide chain, respectively, in order to form sulfhydryl groups and improve its water solubility. And the molecule weight and purity of the modified peptide chain were checked by electron spray ionization mass spectrometry (ESI-MS) and high performance liquid chromatography (HPLC). Then the activated KLH and OVA were conjugated to GA by using  4-(Nmaleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) to form disulfide linkage. In order to obtain artificial antigen of GA, GA was linked to KLH and OVA by sulfo-SMCC coupling at room temperature. The conjugates of KLH-Peptide and OVAPeptide were identified by infrared and ultraviolet spectrophotometry SDS-PAGE and DTNB method, which could prove the activity of the sulfhydryl-groups. In this way, we have obtained a novel artificial immunogen and coating antigen that could be used to raise polyclonal antibody through immunized rabbits.