摘要
目的:构建重组真核表达质粒pEGFP-C1-SR-AⅠ在293T细胞上获得高表达,并对A类Ⅰ型清道夫受体(SR-AⅠ)相应的功能进行鉴定。方法:依据人A类Ⅰ型清道夫受体基因MSR1的cDNA设计引物,用PCR等分子克隆技术构建重组真核表达载体pEGFP-C1-SR-AⅠ,测序证实后,用脂质体法转染293T细胞。用RT-PCR和Western blotting检测SR-AⅠ受体的表达,脂质油红O化学染色法检测实验组细胞的脂质摄取量的变化,通过细胞黏附性实验检测其黏附性的改变。结果:转染后实验组SR-AⅠmRNA表达及蛋白合成明显高于对照组和空载体组,同时发现泡沫细胞转化率由对照组和空载体组的10%显著增加到实验组的20%,且细胞黏附性也提高2倍以上。结论:成功构建了重组真核表达质粒,并在293T细胞获得了高表达,提高了293T细胞的摄脂能力和黏附性,为进一步研究此受体功能的改变对动脉粥样硬化病理发生过程的影响奠定基础。
AIM： To construct the recombinant eukaryotic expression plasmid pEGFP - C1 - SR - AⅠ for the high expression in 293T cells in order to identify functions of savenger receptor - AⅠ （SR - A）. METHODS： The primer was designed according to MSRI cDNA and pEGFP - C1 - SR - AⅠ was constructed by standard molecular cloning technique and enzyme digestion. After sequencing, the plasmid was transfected into 293T cells by lipidosome method. The expression of scavenger receptor - AⅠ was identified by RT - PCR and Western blotting. The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining. Cell adhesion was analyzed by cell adhesion assay. RESULTS ： 24 h after transfection, SR - AⅠ mRNA was highly expressed and the high level of the protein was detected. The ratio of foam cell formation was doubled, the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group. CONCLUSION： The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox - LDL and adhesion in 293T cells by overexpression of SR - AⅠ.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第10期1937-1942,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30671969)
