hURAT1基因真核表达重组体的构建及意义

CONSTRUCTION OF EUKARYOTIC EXPRESSION RECOMBINANT PLASMID PCDNA3.1(-)/hURAT1

目的构建pcDNA3.1(-)/hURAT1真核表达重组体。方法提取人全血基因组DNA,进行PCR扩增,将纯化的扩增产物与表达载体pcDNA3.1(-)连接后转化DH5α感受态细胞。重组质粒经BamHⅠ和EcoRⅠ酶切后,应用核苷酸序列测定方法进行鉴定。结果PCR扩增得到hURAT1第2外显子至第5内含子长1 958 bp的基因片段。该片段与表达载体pcDNA3.1(-)连接后转化DH5α感受态细胞。重组质粒经酶切和核苷酸序列测定方法鉴定,证实hURAT 1片段插入序列和方向正确。结论hURAT 1基因组DNA片段插入真核表达载体pcDNA3.1(-)的序列和方向正确,可用于后续基因功能研究,尤其是编码序列和内含子序列变异的功能研究。

Objective To construct the recombinant eukaryotic expression vector pcDNA3.1 （-）/hURAT1. Methods Total genome DNA was prepared from normal human total blood. DNA was then subjected to PCR amplification. PCR product was cloned into pcDNA3. 1 （-） and then the recombinant vector transformed into DH5a Ecoli. Constructed pcDNA3.1 （-）/hURAT1 was identified by restricting enzyme digestion analysis and DNA sequencing. Results A 1 958 bp fragment, including exon 2 to intron 5 of hURAT1 genome DNA, was obtained by PCR amplification. BamH I and EcoR Ⅰ restriction enzyme digestion and DNA sequencing confirmed with the correct sequence and direction. The pcDNA3.1 （-）/hURAT1 had been successfully constructed. Conclusion The fragment of hURAT1 with correct sequence and direction is cloned into the eukaryotic expression vector pcDNA3. 1（-）, and it can be applied to gene function research in the future, especially to the mutation function research in the CDS or intron.