VEGF-C真核表达质粒的构建及其真核表达

The Combination of VEGF-C Expressing Vector and the Detection of Expression in Tca8113 Cell

目的 研究人血管内皮生长因子 C(VEGF- C)基因功能片段的表达功能及表达活性。方法 以前期从人舌癌克隆得到的 VEGF- C功能片段 c DNA和真核表达质粒 pc DNA3.1(+)构建重组质粒 ,以阳离子脂质体转染法将其导入舌鳞癌细胞 Tca8113,并检测其表达、分泌功能及体外激活能力。结果 通过将长度约为 110 0 bp的VEGF- C功能片段插入 pc DNA3.1(+)的 Eco R 和 Xho 酶切位点之间 ,成功构建出 pc DNA3.1(+) - VEGF- C重组质粒 ,该质粒转染 Tca8113细胞后得到 VEGF- C高表达的舌癌细胞 Vc Tca,转染后的细胞可表达相对分子质量为 5 3× 10 3和 2 9× 10 3的 VEGF- C分子 ,在细胞培养液中可检测到相对分子质量为 2 9× 10 3的 VEGF- C片断。结论 构建的 VEGF- C重组质粒可在真核细胞实现表达 。

Objective To investigate the functional expression of a truncated VEGF-C gene in mammalian cells. Methods A truncated VEGF-C cDNA (with 5′-terminal cleaved between residues 207/208) was used to construct a pcDNA3.1(+)-VEGF-C recombined plasmid, which was cloned into a lingual SCC cell line (Tca8113) by lipid-mediated transfection. The expression of VEGF-C was detected by Western blotting and immunohistochemical staining. Results The VEGF-C fragment was successfully ligated into the pcDNA3.1(+) between EcoRⅠ and XhoⅠ multiclone sites. By Western blotting, the relative molecular mass 53×10 3 and 29×10 3 forms of VEGF-C were detected in cell lysates of VEGF-C transfected cells (VcTca), while the relative molecular mass 29×10 3 form of VEGF-C presented the only band in culture media. According to immunohistochemistry, the VcTca cells showed intensive cytoplasm positive reaction to VEGF-C, whereas faint reaction was found in non-transfected cells and pcDNA3.1(+) mock transfected cells. Conclusion The results indicated that the truncated VEGF-C cDNA was sufficient for expression, secretion and activation when it was transfected into Tca8113 cell.