Study Of Membrane Surface Receptors Of Peptides Specific Binding Macaque Embryonic Stem Cells

Embryonic Stem (ES) cells have unique capabilities to self-renew, growindefinitely, and differentiate or develop into multiple types of cells and tissues. Thekey problem applying ES cells into clinic field is how to isolated ES cells from othercell types. As the “ID” of the cells, marker genes play an important role to recognizeand identify embryonic stem cells.The specific peptide genes of rhesus monkey embryonic stem cells were ligated tothe pGEX-KG expression vector so that they produced the fusion protein GST-peptides.Successfully I have been constructed stable GST expression system and establishedstable GST-peptide purification system. There had many protein sequence informationof cell membrane receptors on RS366.4.A novel method was established in constructing the GST-peptide expression vector.Based on oligonucleotide synthesis technology and competitive ligation principle, theconstruction of five GST-peptide expression vectors were successful, respectively,p-APW, p-HGE, p-GYP, p-TPL, p-SLH. In exploring expression conditions, theoptimal induction time was four hours, and these fusion proteins were highly soluble.Finally, Western blot showed that these fusion proteins were GST tagged proteins.GST-peptides were as bait proteins in subsequent trials.At the same time, I cultured approximately2.0x107RS366.4rhesus monkeyembryonic stem cells. The results of Alkaline phosphatase detection and RT-PCRshowed a strong positive in these cells, indicating that these cells were undifferentiatedstate. Finally, under mild conditions membrane proteins of these cells were extracted, astarget proteins for subsequent experiments.The interaction between the GST-peptides and RS366.4, PMEF membrane proteinswere studied by GST pull-down. And SDS-PAGE analysis revealed that GST-APW,GST-HGE interacted with the specific membrane protein of R366.4, but not PMEF.MALDI-TOF-MS sequencing and analysis showed in these possible proteins whichinteracted with GST-APW, both opalin isoform c [Homo sapiens] and PREDICTED:opalin-like [Callithrix jacchus] are homologous proteins and transmembrane proteins. In the possible proteins which interacted with GST-HGE, unnamed protein product[Macaca fascicularis] is an unnamed protein and Macaca protein, it is likely GST-HGEmembrane receptor molecule.