HeLa细胞表达分泌重组eGFP-DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1/DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP-DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120 KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子/早胚上定位提供了可行的办法。

LOCALIZATION OF EGFP-DPF-1 EXPRESSED AND SECRETED BY HELA CELLS IN OOCYTES

The eukaryotic expression plasmid pEGFP-N1/DPF-1 was constructed through fusing rabbit oviduct-specific glycoprotein (DPF-1) gene to the 5' terminus of eGFP gene. After transfecting the recombinant plasmid into HeLa cells, we got some cell strains expressing and secreting eGFP-DPF-1 stably. The apparent molecular weight of the fusion protein was up to 120 KD indicating the fusion protein was modified. Rabbit oocyte-cumulus cells complexes (COC), the cumulus cells-deprived oocytes or oviductal oocytes were co-cultured with the cell strain or cultured in the conditioned medium derived from the cell strain to observe the distribution of DPF-1 in oocytes after incubation. The results revealed that DPF-1 but not GFP alone associated with zona pellucidae (ZP) of oocytes, especially in the inner layer of ZP, and was evenly distributed in dot-like shape on the outer surface of the cytoplas-mic membrane. In contrast, the cumulus cells around the oocyte did not interfere the association of DPF-1 with oocyte, and no trace of DPF-1 was found in the cumulus cells. The experiment proved that the oviductin expressed and secreted by eukaryotic cells in vitro could bind to oocytes, and was directly observed by using green fluorescent protein as a tracer. These results provided some interesting informations for the further studying on the functions of the oviductin.