Screening And Identification The Receptor Of Anticancer Hexapeptide In Human Ovarian Carcinoma Cells

Objective To screen and identify the receptor of anticancer hexapeptide derived in bata casein in human ovarian cancer cell lines (SKOV3) by pull-down and two-dimensional electrophoresis.Methods According to its sequence, PGPIPN gene was designed and synthetized with the restriction sites of BamHI/XhoI, then was linked with the expression plasmid vector, pGEX-4T-1. This expression plasmid vector was transformed into E.coli, BL21. The GST-tagged fusion protein as the bait protein was expressed in BL21by IPTG inducing at low temperature. The total proteins were extracted from SKOV3as the capture protein. By GST pull-down, the target protein was captured and screened from the above extracted proteins. To ensure the accuracy of the screening results, using two-dimensional electrophoresis to further identify, which use the total protein extracted from SKOV3cells as samples.By SDS-PAGE and two-dimensional electrophoresis, the target protein was identified with PGPIPN labelled fluorescein isothiocyanate (FITC).Results The two protein bands were seen on SDS-PAGE gel, one being the bait protein and another being the target protein which was identified by immunofluorescence microscopy with the fluorescence labelled peptides. There are two fluorescent binding sites which were identified under immunofluorescence microscopy with the fluorescence labelled peptides.A preliminary analysis of the bait protein and the phosphor dots by electrospray mass spectrometry, identified four functions, similar to the nature of the associated proteins which were aldolase A, aldolase C,eukaryotic translation elongation factor1delta isoform2,60S ribosomal protein L5and similar to F-box only protein2. Conclusion The receptor of anticancer hexapeptide derived in bata casein was screened in human ovarian cancer cell lines (SKOV3) by pull-down and two-dimensional electrophoresis. This study will lay the foundation for understanding the anticancer mechanism and transduction pathways of PGPIPN.