重组人EGFL6在HEK293细胞中的瞬时表达

Transient Expression of Human EGFL6 in HEK293 Cells

旨在通过构建EGFL6的真核表达载体,在HEK293T细胞中进行表达,并利用HEK293T细胞表达的EGFL6条件培养基,探讨EGFL6对人黑色素瘤A375细胞和人卵巢癌SKOV3细胞增殖的影响。利用PCR方法亚克隆得到的EGFL6基因片段及EGFP基因片段,用重叠PCR方法构建成EGFP-EGFL6融合基因片段,并分别将测序正确的EGFP-EGFL6融合基因片段及EGFP基因片段构建到真核表达载体pAAV中。表达载体质粒去内毒素纯化后,瞬时转染HEK293T细胞进行表达,荧光定位分析和Western blot检测EGFL6的表达。CCK-8试验分析含EGFP-EGFL6融合蛋白的HEK293T条件培养基对A375细胞和SKOV3细胞的增殖能力的影响。结果显示,酶切鉴定pAAV-EGFP-EGFL6和pAAV-EGFP两个重组表达载体均构建成功;EGFP荧光定位及Western blot结果均显示EGFP-EGFL6在HEK293T细胞中成功表达;增殖抑制试验结果显示EGFL6促进了A375和SKOV3细胞的增殖,对肿瘤细胞的促增殖率分别达到(37.79±14.05)%和(30.53±6.31)%。成功地将EGFP应用于EGFL6真核表达载体的构建与表达,结果表明人EGFL6促进了A375和SKOV3细胞的增殖。

To construct eukaryotic expression vector of EGFL6 and transient transform HEK293T cell to investigate EGFL6 effects on tumor cells with EGFL6 conditioned medium.EGFL6 gene fragments and enhanced green fluorescence protein（EGFP）gene fragments were sub-cloned by PCR and combined to construct EGFP-EGFL6 fusion protein gene fragment by overlapping PCR.The sequencing identified EGFP gene fragments and EGFP-EGFL6 gene fragments were inserted to eukaryotic expression vector pAAV to construct the expression vectors.Purified by endotoxin-free kit,the expression vector plasmids were transiently transformed into HEK293T cells.The expression of EGFL6 fusion protein was identified by fluorescence localization and Westernblot analysis.The effects of EGFL6 on tumor cell proliferation of A375 and SKOV3 were assayed by CCK-8 reagent using EGFL6 expression conditioned medium.The restriction enzyme assay showed that the expression vectors of pAAV-EGFP and pAAV-EGFP-EGFL6 were constructed successfully.EGFP fluorescence localization and western blot assay results showed that EGFP-EGFL6 fusion protein was expressed successfully in HEK293T cells.The cell proliferation assay showed that EGFL6 enhanced tumor cell proliferation of A375 and SKOV3 and the cell proliferation rates reached about（37.79±14.05）% and（30.53±6.31）%.EGFL6 was expressed in HEK293T cells and the EGFL6 expression conditioned medium could promote tumor cell proliferation.