Preparation Of Polyclonal Antibody, Tissue Expression And Co-localization With IGFBP-3 Of GalNAc-T14

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous N-acetylgalactosaminyltransferases which transfer GalNAc to serine and threonine acceptor sites. Recent studies demonstrate that O-glycosylation may be closely related to the carcinogenesis and development of tumours. Hence, Studys on the N-acetylgalactosaminyltransferases family are getting more and more attention. Polypeptide N-acetylgalactosaminyltransferases 14 (GalNAc-T14) is a new member of glycosyltransferases family. It isn't getting more attention, so we need research deeply the expression and function of GalNAc-T14 in cancer cells and tumor tissues.The function of GalNAc-T14 was studied from three aspects as follows:1 The sequence of GalNAc-T14 was analyzed using bioinformatics. The cDNA of interest was subcloned into a procaryosis expression vector pET-DsbA to construct pET-DsbA/ GalNAc-T14s, then expressed by fusion gene with His-tag. The expression of recombinant fusion protein was better induced by IPTG,then better purified by Ni-IDA Resin. Polyclonal antibody against GalNAc-T14 was prepared from immuned New Zealand white rabbit. The titer and specificity of the prepared antibody were analyzed by immunodiffusion and western blot. The expression of GalNAc-T14 in tumor tissues was dectected by western blot. The result indicates that polyclonal antibody against GalNAc-T14 is high specificity,and if the low expression of GalNAc-T14 in a tissue is high differentiating malignancy and Poor Outcome.2 The cDNA was subcloned into a eukaryotic expression vector pEGFP-N2 to construct pEGFP-C1/GalNAc-T14. Human breast carcinoma cell line MCF-7 and human renal cell carcinoma (RCC) 786-O cells were transfected with pEGFP-C1/GalNAc-T14 using lipofectamine 2000. Subcellular localization was observated by immunofluorescence which using IGFBP-3 antibody. The result indicates that the co-localization of GalNAc-T14 and IGFBP-3 exist.3 The cDNA was subcloned into a eukaryotic expression vector pMT/BIP/V5-HIS C to construct pMT/BIP/V5-HIS C/GalNAc-T14. The recombinant plasmid was transfered into Drosophila cell line S2. A great deal of GalNAc-T14 protein is expressed. It will be used for future.Research and discuss the function of GalNAc-T14, Our findings may provide the next step for further study, and may be very important to offer the materials and basis.