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本研究旨在建立表达重组野生型和突变型血小板膜糖蛋白GPIb-Ⅸ复合物的中国仓鼠卵巢(Chinese Hamster Ovary,CHO)细胞研究模型,为进行GPIb-Ⅸ生理功能的相关研究奠定基础。从表达野生型或缺失突变型GPIbα的大肠杆菌宿主菌中抽提质粒并用EcoRI酶切鉴定,然后将分别表达GPIbα、GPIbβ和GPIX3种基因的质粒共转染到中国仓鼠卵巢细胞中,通过免疫共沉淀、Western blot和流式细胞仪检测其表达效果。结果表明,转染48小时后,CHO细胞裂解物和细胞表面均可检测到GPIb-Ⅸ复合物的表达。结论:成功构建了表达血小板膜糖蛋白GPIb-Ⅸ复合物的CHO细胞研究模型。
The aim of this study was to establish a Chinese Hamster Ovary (CHO) cell model that expresses recombinant GPIb-Ⅸ complex of wild-type and mutant platelet glycoproteins, and lay the foundation for further studies on the physiological functions of GPIb-Ⅸ. Plasmids were extracted from E. coli host strains expressing wild-type or mutant-type GPIbα and digested with EcoRI. Plasmids expressing GPIbα, GPIbβ and GPIX genes were co-transfected into Chinese hamster ovary cells. Precipitation, Western blot and flow cytometry to detect the expression effect. The results showed that the expression of GPIb-Ⅸ complex was detected on both lysates and cell surfaces of CHO cells 48 hours after transfection. Conclusion: The CHO cell model of GPIb-Ⅸ complex expressing platelet glycoprotein was constructed successfully.