抑癌基因肌足蛋白抗体制备及其在肝细胞癌中的检测

Preparation of an anti-myopodin antibody and detection of myopodin protein in hepatocellular carcinoma

目的制备肌足蛋白(myopodin)抗体,探讨myopodin在培养细胞以及在肝癌组织中的表达。方法构建pGEX4T2-N-myopodin重组表达质粒,转化大肠杆菌并诱导蛋白表达,N-myopodin蛋白纯化,免疫新西兰家兔得到多克隆抗体。对HeLa、T24、MG63、HEK293T和NIH3T3等细胞株进行myopodin免疫荧光染色,并对原发性肝细胞癌组织和癌周正常肝组织进行myopodin蛋白印迹检测。结果兔抗N-myopodin多克隆抗体具有良好的特异性和敏感性。免疫荧光染色发现,不同细胞株细胞内myopodin的分布不同。蛋白印迹检测显示,myopodin在肝癌组织表达低于癌旁组织。结论myopodin在细胞内分布不同,myopodin蛋白表达与肝细胞癌发生有一定关联。

Objective To prepare anti-myopodin antibody and study the significance of myopodin expression in cell lines and hepatocellular carcinoma tissues. Methods Construction of the plasmid pGEX-4T-2-N-myopodin, flowed by transformed and induced it in E. coli, than expression and purification of N-myopodin protein. Preparation of rabbit anti-myopodin antibody. The expression of myopodin was analyzed by immunofluorescence and Western blot in HeLa, T24, MG63, HEK293T and NIH3T3 cell lines and hepatocellular carcinoma tissues and normal liver tissues respectively. Results The anti-myopodin antibody was proved having good specificity and sensitivity. Immunofluorescence staining showing that the subcellular distribution of myopodin was cell type-dependent. The myopodin expression level of hepatocellular carcinoma tissues was lower than that in normal liver tissues. Conclusion The subcellular distribution of myopodin is cell type-dependent. There is a correlation between myopodin protein and development of hepatocellular carcinoma.