[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 cytoplasmic 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.
Luo
Jin Ping
JP
National Key Laboratory of Contraceptives and Devices Research, Shanghai Institute of Planned Parenthood Research, Shanghai Medical College, Fudan University, Shanghai 200032.
Yan
Gui Jun
GJ
Gu
Zheng
Z
Tso
Jia Ke
JK
D000818:Animals; D001665:Binding Sites; D005260:Female; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; C074599:oviductin