VEGF-C真核表达质粒的构建及其真核表达

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 ,并能被分泌和激活

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

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 EcoR I and Xho I multiclone sites. By Western blotting, the relative molecular mass 53 x 10(3) and 29 x 10(3) forms of VEGF-C were detected in cell lysates of VEGF-C transfected cells (VcTca), while the relative molecular mass 29 x 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.