Preliminary Study On The Interaction Of PRMT2,ER-?36 And ER-?66 In Vitro

Obejecive: This study aimed to construct the prokaryotic expression vectors of pET-28a-PRMT2,p GEX-6p-1-ER-?36,p GEX-6p-1-ER-?66,then to investigate the relationship of PRMT2 and ER-?36 and ER-?66 in vitro,to provided the basis and direction for the effect of PRMT2 on the proliferation and drug resistance of breast cancer cells.Method: 1.construction and identification of the prokaryotic expression vectors: The corresponding gene primer was designed according to the target gene,which was selected from the gene library.And the target gene was amplified by PCR.Double enzyme digestion,ligation experiment,transformation experiment,recombinant plasmid amplification and extraction was used to construct the prokaryotic expression vectors.Then they were detected by double enzyme digestion and gene sequencing.2 expression of the prokaryotic vectors and purification of the protein: The prokaryotic expression vectors of pET-28a-PRMT2,p GEX-6p-1-ER-?36,p GEX-6p-1-ER-?66 were transformed into Bl21-de3-expressing Escherichia coli.And low-dose experiment was performed to test.Then the protein was detected by western blot,which was expanded and purified.3.GST-pull down experiment: The relationship of PRMT2,ER-?36 and ER-?66 was analyzed by GST-pull down in vitro.GST-ER-?36,GST-ER-?66,GST,His-Tag-PRMT2 and Glutathione agarose beads were incubated and repeatedly washed.The expression of PRMT2,ER-?36,ER-?66 and GST was analyzed by the western blot.Results:1.Construction and identification of the prokaryotic expression vectors: pET-28a-PRMT2,p GEX-6p-1-ER-?36,p GEX-6p-1-ER-?66 of prokaryotic expression vectors were constructed successfully,which was then identified by double enzyme digestion that suggested the obtained gene fragment were consistent with the expected.Also the construction sequence was consisted to the target gene base sequence that is identified by gene sequencing,which showed the prokaryotic expression vectors were established successfully.2.Expression of the prokaryotic vector and purification of the protein: The prokaryotic expression vectors of pET-28a-PRMT2,p GEX-6p-1-ER-?36,p GEX-6p-1-ER-?66 were transformed into Bl21-de3-expressing Escherichia coli.And the target proteins His-Tag-PRMT2,GST-ER-?36,GST-ER-?66 and GST were obtained.Their molecular weight consistent with predicted protein,which were no obvious bandage.3.GST-pull down experiment: The GST fusion protein ER-?36,ER-?66 and GST were immobilized on Glutathione agarose beads and interacted with PRMT2,which was analyzed by the Western blot.The result showed the relationship between PRMT2 and ER-?36 and ER-?66 in vitro.PRMT2 and ER-?36 and ER-?66 were expressed,but the GST did not interact with PRMT2.Conclusion:The pET-28a-PRMT2,p GEX-6p-1-ER-?36,p GEX-6p-1-ER-?66 of prokaryotic expression vectors were constructed successfully.And we confirmed the interaction among PRMT2 and ER-?36,ER-?66 in vitro.