人MCHR2基因shRNA真核表达载体构建及鉴定

Construction and identification of eukaryotic expression vectors expressing shRNA sections targeting human MCHR2

目的构建针对人黑色素浓集激素受体2(MCHR2)的shRNA真核表达载体,并在人胚肾293(HEK293)细胞中观察其对MCHR2基因表达的影响。方法根据MCHR2基因序列,设计并合成特异性的小干扰片段,并将其定向克隆到带有卡那霉素抗性和增强绿色荧光蛋白的真核表达载体pGenesil-1中,对重组质粒进行酶切分析和DNA序列测定。用脂质体将重组质粒转染到HEK293细胞中,观察其在mRNA和蛋白水平对MCHR2的影响。结果通过酶切鉴定和序列测定,成功构建四条表达短发夹RNA的质粒及其阴性对照质粒,并成功转染到HEK293细胞株中。四条shRNA真核表达载体在mRNA水平对MCHR2抑制率分别为54.9%、66.4%、56.9%、45.8%;在蛋白水平对MCHR2抑制率分别为62.0%、81.0%、73.3%、44.2%。结论针对人MCHR2的shRNA真核表达载体成功构建及鉴定为进一步研究MCHR2功能奠定了良好的基础。

Objective To construct eukaryotic expression vectors expressing short hairpin RNA（shRNA）sections targeting human MCHR2 and to observe their effects on MCHR2 gene expression in HEK293 cell ling Methods According to the sequence of human MCHR2 gene, the oligonucleotides of shRNA were designed and synthesized and directionally cloned into plasmid pGenesil-1 with enhancing green fluorescence protein（EGFP）gene and Kan gene. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into HEK293 cell line by Lipofectamine^TM2000, the effects on MCHR2 at mRNA and protein levels were observed. Results Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into HEK293 cell successfully. MCHR2 transcript was reduced by about 45. 8%-66. 4%, the protein of MCHR2 was reduced by about 44.2%-81.0% in four transfectants respectively. Conclusion The construction of eukaryotic expression vectors ex pressing shRNA sections targeting human MCHR2 and identification successfully established a favourable foundation for further study on the function of MCHR2.