Preparation and identification of the rabbit antibody against human sialin

AIM: To prepare the rabbit antibody against human sialin and identify its properties. METHODS: Recombinant expression vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1-38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin (N1-38) fusion protein was expressed in E. coli JM109 at 37 degrees C in the presence of IPTG at 0.1 mmol/L for induction for 3 hours, purified by GSTrap FF, and then used as the immunogen to prepare the rabbit polyclonal antibody. The properties of antiserum against human sialin were identified by ELISA, Western blot and immunocytochemistry. RESULTS: The recombinant expression plasmid pGEX-5X-1-sialin was constructed. The GST-sialin (N1-38) fusion protein was highly expressed with a molecular weight of 30 kDa, and the yield of the fusion protein was about 20% to 30% in total E. coli protein. The titre of antiserum against human sialin was 1:32,000. Western blot analysis proved the rabbit polyclonal antibody could identify both GST-sialin (N1-38) fusion protein and GST. Besides, it specially recognized a 55 kDa band expressed in the human submandibular gland (HSG) cell line. The antigen recognized by the antibody was located in the cytoplasm and nucleus of HSG cell. CONCLUSION: The successful preparation of the polyclonal antibody against human sialin will provide efficient affinity reagent for further functional study of sialin expressed in human salivary glands.