人CIDE3基因克隆和真核表达载体构建及促凋亡作用的研究

The cloning and construction of eukaryotic expression vector of human CIDE3 and its proapoptotic effect on HeLa cells

目的:克隆人诱导细胞死亡的DFF45样效应因子3(CIDE3)全长基因,构建真核表达质粒pcDNA3.l(+)-CIDE3,并检测其促凋亡作用。方法:①提取人腹部皮下白色脂肪组织的总RNA,经RT-PCR获得CIDE3全长基因,克隆入真核表达载体pcDNA3.l(+),构建重组质粒pcDNA3.l(+)-CIDE3,并进行序列测定。②用脂质体法将pcDNA3.l(+)-CIDE3转染人宫颈癌细胞系HeLa细胞,通过TUNEL法检测细胞凋亡情况。结果:①经RT-PCR扩增得到特异性717 bp的目的片段,构建的重组质粒pcDNA3.l(+)-CIDE3经序列测定,结果与Genbank发表的序列完全一致。②转染Hela细胞后,经Tunel染色法检测发现,转染pcDNA3.l(+)-CIDE3后有大约25%的细胞发生了凋亡,与转染空质粒pcDNA3.l(+)(<5%)及未转染质粒的细胞相比,其凋亡细胞明显增多。结论:成功克隆了人CIDE3全长基因,构建了真核表达质粒pcDNA3.l(+)-CIDE3,并验证了CIDE3基因可诱导细胞发生凋亡的生物学作用,为进一步研究CIDE3的生物学功能奠定了坚实基础。

Objective：To clone the human cell death-inducing DFF45-1ike effectors 3 （CIDE3） full length eDNA for construction the eukaryotic expression vector pcDNA3.1 （ ＋ ）-CIDE3 and to study its bioactivity. Methods：（1） Total RNA was extracted from human white adipose tissues and the desired eDNA fragment was obtained by RT-PCR. After the fragment had being inserted into an eukaryotic expression vector pcDNA3.1 （ ＋ ）, the resulted recombinant plasmid pcDNA3.1 （ ＋ ）-CIDE3 was transformed into DH5α. The positive clone was selected and confirmed to contain full length of CIDE3 eDNA by agarose gel and DNA sequence analysis. （2）After the pcDNA3.1（ ＋ ）-CIDE3 plasmid was transfected into HeLa cells under lipofectamine mediation, the effect of target gene expression on growth of HeLacells was analysed by TUNEL staining. Results：（1） The recombinant eukaryotic expression plasmid pcDNA3, 1（ ＋ ）-CIDE3 was constructed successfully and the sequence of CIDE3 was consistent with that of genebank. （2）After transfected pcDNA3, 1（ ＋ ）-CIDE3, HeLa cells presented distinguished apoptosis （about 15% ）, compared with that of transfected plasmid pcDNA3.1（ ＋ ） （ 〈5% ） and untreated（0）. Conclusion：In this study, we have successfully cloned CIDE3 full length cDNA, constructed its eukaryotic expression plasmid pcDNA3, 1（ ＋ ）-CIDE3 and conformed that CIDE3 could induce apoptosis, which provide the foundation for the further study the function of CIDE3.