人MC1R基因在杆状病毒-昆虫细胞表达系统中的表达

Expression of Human MC1R Gene in Baculovirus-Insect Expression System

目的利用杆状病毒表达系统进行人恶性黑色素瘤A375细胞MC1R基因在Sf9昆虫细胞中的表达。方法以pMD18-T-MC1R为模板,利用PCR方法扩增人MC1R基因,将MC1R基因连接到pfastBac1质粒上,与穿梭载体DH10Bac转座,获得Bacmid-MC1R质粒后,通过脂质体介导,转染Sf9细胞,使其表达重组杆状病毒,经SDS-PAGE检测表达产物,放射受体分析法检测目的蛋白MC1R活性。结果Bacmid-MC1R质粒转染Sf9细胞后的SDS-PAGE图谱,在相对分子质量为35000处有一条新生蛋白带。放射受体分析结果显示表达的蛋白与标记配体特异亲和,具有生物学活性。结论MC1R基因成功在真核细胞中得到表达。

Objective To express the MCIR gene of human malignant melanoma A375 cells in insect cell Sf-9 by using baeulovirus expression system.Methods Amplify human MCIR germ by PCR method using pMD18-T-MCIR as a template. Insert the gene to plasmid pfastBael and transpose into a shuttle vector DH10Bac. Transfect Sf9 cells with the obtained Baernid-MCIR plasmid in the mediation of liposome. Identify the expressed product by SDS-PAGE. Detect the target protein MCIR by RRA.Results SDS-PAGE profile showed a new protein band with a relative molecular weight of 35 000. The radioligand binding assay proved that the expressed protein could bind 125 I-MSH. Conclusion MCIR gene was suceessfully expressed in Sf9 cells.