Functional characterization of partial recombinant goat conglutinin: Its role as innate immunity marker and use as antigen in sandwich ELISA

摘要

Conglutinin, a liver synthesized versatile innate immune marker consisting C-type lectin domain belongs to collectin superfamily of proteins. The protein, first detected in bovine serum as soluble pattern recognition receptor (PRR) has wide range of antimicrobial activities. In the present study, open reading frame (ORF) encoding neck and carbohydrate recognition domain (NCRD) of goat conglutinin gene ligated to the vector pRSET A was expressed in E. coli BL-21(pLys) cells. The 27 kDa recombinant protein (rGCGN) purified by single step Ni+2-NTA affinity chromatography was found to cross-react with recombinant anti-buffalo conglutinin antibody raised in poultry. Further, it displayed calcium-dependant sugar binding activity towards yeast mannan and calcium-independent binding activity towards LPS. The mannan binding activity of rGCGN was inhibited in the presence of N-acetyl-glucosamine because of higher affinity towards this sugar. The recombinant protein was found to stimulate production of superoxide ions and hydrogen peroxide in goat neutrophils, which are instrumental in stimulating phagocytic activity of cells. When used as antigen in Sandwich ELISA, straight line (Y = 0.299x + 0.067, R-2 = 0.997) was observed within the concentration range of 200-1000 ng/100 mu l of rGCGN. Using this equation, the native conglutinin concentration in goat sera was estimated to be 0.5-7.5 mu g/ml. The results indicated that prokaryotically expressed functionally active rGCGN can be used as antigen to assess native serum conglutinin levels in Sandwich ELISA and as immunomodulator in therapeutic applications to sequester unwanted immune complexes from the circulation.