人CD46启动子真核表达载体的构建

Construction of Eukaryotic Expression Vectors by Use of Human CD46 Promoter

为了构建人CD46(hCD46)启动子指导目的基因表达的真核表达载体,提取HeLa细胞基因组DNA,用PCR扩增出hCD46基因的启动子区域,序列分析结果表明其与GenBank中hCD46基因5′端某片段的同源性为99.9%。用此启动子替换pcDNA3EGFP中的CMV启动子,并在hCD46启动子和EGFP基因之间插入兔β-球蛋白基因第二内含子(RGI),得到的重组表达载体转染CHO和SP2/0两种鼠源细胞,流式细胞术检测表明CHO细胞EGFP的表达量高于SP2/0细胞,表达特性与人体CD46相似;RGI可以增强EGFP的表达量,但不改变其表达的组织特异性,提示克隆的hCD46启动子可以用于研制模拟人体CD46基因表达特性的转基因小鼠。

To construct eukaryotic expression vectors that utilize human CD46 promoter to drive expression of genes, hCIM6 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.