Cloning And Expression Of Human UDP-Gal: BetaGlcNAc Beta 1,3-galactosyltransferase Polypeptide 7 Gene And Study On The Feature And Role Of Its Encoding Protein

Object : UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase is a subfamily of glycotransferase family, its β3Gal-T activities widely eixst in animals. This thesis aims to clone a novel member of the β3GalTs family and do some study on the gene structure and role on nuclear and protein levels. Methods: 1. Obtain β3GalT7 cDNA sequence by means of electric cloning , isolate it from some human cDNA library(lung, colon, Clontech Co. ltd.), insert it into vector pGEM-T and verify by sequencing. Analysis the gene and encoding protein with biological sofewares, web services and databases. The subcellular location was observed using fluoroimmunocytochemistry. 2. Use semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Northern blotting to detect the mRNA expression discrepancy of β3GalT7 in different cells and tissues. 3. The cDNA gene encoding β3GalT7 was subcloned from pGEM-T into prokaryotic expression vector pGEX-6p-1, transform the recombinant plasmid into Escherichia coli B21, IPTG was used to induce the expression of the target gene; The polyclonal antiserum was prepared by immunizing rabit with purified β3GalT7 protein; Detect the protein expression discrepancy of β3GalT7 in different cells and tissues by Western Blot and immunochemistry. Results: We isolated a novel member of UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase family from a human lung cDNA library and named it β3GalT7(AY277592, EC2.4.1.). β3GalT7 is mapped to chromosome 19q13.2. It contains an ORP with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its moledular weight and isoeledtric point are predicted to be 43.3kDa and 8.66 respectively. The expression pattern of P3GalT7 wasmainly in cytoplasm around nuclear membrane. The molecular weight of protein when expressed in E.coli corresponded to that expected. Northern blotting showed that p3GalT7 was highly expressed in lung, throat and ileum, wehereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that p3GalT7 is differentially transcribed in human tumor cell lines. Western blottling showed that the enzyme was highly expressed in many tumor cell lines. Immunochemistry analysis showed the enzyme was highly expressed in some tumor tissues and indicated an expression discrepancy between tumor tissues and normal tissues while the later showed a relatively low expression. Conclusion: We isolated a novel member of UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase family successfully and named it P3GalT7. We successfully expressed the |33GalT7 gene in Escherichia coli BL21 and obtained anti-p3GalT7 polyclonal antiserum, which could be used in studing the role of p3GalT7. Assay in both nuclear and protein levels showed that the expression of P3GalT7 altered differently in different cell lines and tissues, which may due to a substrate specificity. The result that the enzyme was relatively highly expressed in some tumor tissues than in nomal tissues may suggest that p3GalT7 is involved in the occurrence and development of tumor biological behavior.