Molecular Cloning and Functional Characterization of the 5′-Flanking Region of Human Intestine Tissue-specific Antigen A33 Gene

With the genome walking method, A 940 bp of 5′ flanking region of the human intestine tissue\|specific antigen A33 gene was cloned from the Eco R Ⅴ library. The identity was confirmed by PCR with genomic DNA as template. As expected, the PCR product confirmed that the 940 bp region was the 5′ flanking region of the human intestine tissue\|specific antigen A33 gene. The Accession Number is AF200626 in GenBank. The transcription start site of human A33 gene was identified to be 10 nucleotides downstream from the TATA box by primer extension analysis. The EGFP reporter system to scan regulatory elements in the 5′ flanking region of the human intestine A33 gene was used. A series of plasmids in which various extents of A33 promoter were placed upstream of the EGFP in pEGFP 1 vector was constructed. Transfecting the recombinant vectors into LoVo, HeLa and 293 cells and comparing fluorescence intensity among these four cell lines, it was found that the -104～+25 bp region had almost full activity of the promoter with a tissue specific expression manner.