HAb18G/CD147真核荧光蛋白的表达及鉴定

The Construction and Expression of Eukaryotic EGFP Expression Vector Containing Hepatoma Assosciated Antigen HAb18G/CD147

构建肝癌相关抗原HAb18G/CD147全长及缺失片段E51(第22—50位氨基酸缺失)的真核荧光蛋白表达载体,进一步研究该基因在肝细胞肝癌发生中的作用。通过PCR及OverlappingPCR的方法获得目的基因,与荧光载体pEGFP-N1双酶切、连接、转化E.coliJM109,构建含有肝癌相关抗原HAb18G/CD147全长及E51的真核荧光蛋白表达载体。并经限制性酶切及序列分析证明插入是否正确。用阳离子脂质体介导转染COS-7细胞,进行瞬时表达;荧光显微镜观察EGFP表达,通过流式细胞术检测蛋白的表达情况,明胶酶谱法鉴定其功能。成功地构建了真核荧光蛋白表达载体pEGFP-N1/HAb18G、pEGFP-N1/E51,并经限制性酶切及序列分析证明外源基因插入正确;流式细胞术鉴定该蛋白表达正确;功能实验证实缺失片段E51不具有诱导成纤维细胞分泌MMPs的功能。结果显示HAb18G/CD147基因第22—50位氨基酸与其刺激成纤细胞分泌MMP和功能有密切关系。实验结果为HAb18G蛋白分子的生物学功能研究奠定了基础。

The DNA fragments encoding HAb18G/CD147 and truncated fragment E51 （deleting amino acid residue 22--50） were amplified by PCR. Then PCR products were digested with BamHI and XhoI and ligated into eukaryotic expression vector pEGFP-N1. The recombinant plasmids mixed with lipofactamine2000 were transiently transformed into COS-7 cells. The results demonstrated that the products generated by PCR were inserted to vector pEGFP-N1 correctly, which were identified with digestion of restrict endonuleases and were sequenced. The expression of HAb18G/CD147 and E51 were detected by fluorescence microscope and flow cytometry assay. Gelatin zymography analysis was carried out by using transfected COS-7 cells containing HAb18G/CD147 and E51 cocuhured with human dermal fibroblasts, respectively. The results showed that the secretion and activation of MMPs was only observed in cells expressing HAb18G/CD147 not in E51. It suggested that amino acid residues 22--50 of HAb18G/CD147 may be critical to the MMPs secretion and HCC progress and invasion, therefore, these amino acid residues may become potential target for cancer therapy.