大鼠胰岛素Ⅱ基因启动子克隆与功能验证

Cloning and functional verification of rat insulin 2 gene promoter

【目的】克隆大鼠胰岛素II基因启动子(RIP2)序列并对其功能进行验证,为培育胰岛特异性表达外源基因的转基因动物奠定基础。【方法】根据Gen Bank中已发表的RIP2序列(J00748.1)设计引物,以大鼠基因组DNA为模板,PCR扩增RIP2序列,连接p MD18-T载体后用Hind III和Bam H I进行双酶切,回收RIP2片段并连接至不含启动子的p EGFP-1载体上构建真核表达载体p EGFP-1-RIP2。重组质粒转染PK15细胞,24 h后观察细胞荧光蛋白表达情况。【结果】克隆获得的RIP2序列与参考序列(J00748.1)的同源性为99.9%,存在3处碱基差异,即从起始密码子ATG(+1)上游-57处有一个G碱基缺失,-387处C突变为A,-707处插入一个G碱基。RIP2序列存在一个TATA-box(-206^-201位点)和一个CAAT-box(-343^-339位点)。以重组质粒p EGFP-1-RIP2转染PK15细胞,24 h后能观察到绿色荧光。【结论】克隆获得的大鼠RIP2序列具有启动子功能,但活性较CMV启动子弱。

【Objective】Cloning and functional verification of Rat insulin 2 promoter（RIP2） were conducted to lay the foundation for breeding transgenic animals of specific expression target genes of insulin. 【Method】According to the RIP2sequence（No. J00748.1） was published in Gen Bank, a pair of primer was designed to amplify the RIP2 sequence by PCR method. The RIP2 sequence was connected to the p MD18-T vector, the restructuring vector was doubly digested by Hind III and Bam H I, and then the recycling RIP2 sequence was connected to p EGFP-1 vector to construct the p EGFP-1-RIP2 expression vector. The constructing vector was transfected into PK15 cells, and fluorescent protein expression in cells was observed after 24 hours. 【Result】The cloned RIP2 sequence was 703 bp（-871--169 sites） in length and had 99.9% of homology the reference sequence（No. J00748.1）. There existed 3 base mutations, from initiation codon ATG upstream（＋1）, G base deletion was at-57 site, C/A mutation at-387 site, G base insertion at-707 site. The RIP2 sequence contained 2 promoter elements： TATA-box（-206--201 sites） and CAAT-box（-343--339 sites）. Green fluorescence was observed from the PK15 cells transfected p EGFP-1-RIP2 plasmids after 24 hours. 【Conclusion 】The cloned RIP2 sequence has promoter function, but its activity is weaker than CMV promoter.