中国生物工程杂志

To construct eukaryotic expression vectors that utilize human CD46 promoter to drive expression of genes, hCD46 promoter was amplified by PCR using genomic DNA from HeLa cells as template. The PCR product of 1.5 kb was subcloned into pMD18-T vector and submitted to sequence analysis. Nucleotide sequence alignment showed that the self-cloned hCD46 promoter was 99.9% homologous with a DNA fragment from the 5’ end of hCD46 gene published in 2006, differing only in 2 nucleotides. The hCD46 promoter was used to replace the CMV early promoter in pcDNA3EGFP; and the rabbit β-globulin gene intron 2 (RGI) was inserted between hCD46 promoter and EGFP gene in order to enhance the expression level of the gene of interest. The recombinant expression vectors, pCDPEGFP and pCDPEGFP-RGI, were transfected into CHO and SP2/0 cells, respectively. Detection by FACS revealed that the expression level of EGFP in transfected CHO cells was higher than that in transfected SP2/0 cells, similarly to the expression property of hCD46 in vivo in human. RGI could enhance the expression level of EGFP in each cell line without altering the cell line-specific expression characterization. These data indicate that the eukaryotic expression vectors containing human CD46 promoter could be suitable for the development of transgenic mice that mimic the expression properties of hCD46.