The Purification And Characterizition Of Human PTP-MEG2 And The Preparation Of Its Polyclonal Antibody

MEG2 protein tyrosine phosphatase (PTP-MEG2), which is PTP megacaryocyte1 protein tyrosine kinase (PTP) subfamily members of the family also includes PTPH1, PTPBAS and PTPD1.Its open reading frame encoding a 593 amino acids and contains molecular weight of 68 kDa protein,It did not clear signal and transmembrane sequence, PTP for its C-terminal catalytic domain, and other known PTP is 30-40% homology;N-terminal 250 amino acids, and the cells within the retinal uncoupling protein 28% homology with the SEC 14p yeast protein with 24% identity,Yeast SEC14p protein phosphatidylinositol transfer activity and is adopted by the yeast Golgi protein secretion of the necessary role of the protein, which may be related to the Gorky PTP-MEG2 of the function.PTP-MEG2 reorganization of the protein can be expressed in E. coli and soluble PTP vitality.PTP-MEG2 mRNA in the different organs of the human body from 12 kinds of cells can be detected, it also shows that such a broad expression of PTP.PTP-MEG2 hinted that the structure of this enzyme may be involved in the tyrosine phosphorylation of the hydrophobic ligand and the transfer of the functions of Gorky.Of polycythemia vera (PV) is a myeloproliferative disease, accompanied by red blood cells, myeloid blood cells and the excessive proliferation.Previously, confirmed from the experimental PV patients erythroid colony-forming cells (ECFC) containing an activity over the coupling membrane tyrosine phosphatase now confirm that this enzyme is PTP-MEG2, with a structure containing lipid within the domain of the cell.PTP-MEG2 in the PV cells because of its excessive activation of the membrane fraction on the increase in the distribution of the.With the development of a mature ECFC RBC, PTP-MEG2 protein level is gradually decreasing, but the coupling membrane PTP-MEG2 in PV cells has lasted a long time, and enhanced cell colony-forming ability.PTP-MEG2 dominant negative mutant expression in vitro in both inhibited the growth and expansion of ECFC. This shows that PTP-MEG2 erythroid cells in the development process plays an important role, for the development of drugs to reduce the proliferation of red blood cells provides a good therapeutic targets.Main results are as follows:1. PTP-MEG2 catalytic domain of the gene cloning and expression of△MEG2.We use pBluescript II KS MEG2 as a template, which was amplified by PCR, and shert its N-terminal and C-terminal into EcoRI restriction endonuclease sites and the suspension of codon, DNA electrophoresis results shows that the PCR product sizes are correct. PCR amplification will be the PTP-MEG2 gene, and we connect it into pT7 expression vector, and then digested with EcoR I, the DNA electrophoresis identification shows the correct size. Through recombinant DNA sequencing proved the correct sequence, recombinant DNA expression vector will be amplified into E. coli Rosetta DE3, access to the strain and high expression of a highly efficient expression.2. Purification and Characterization of PTP-MEG2(1): Extract of target protein extract: Put the bacterium in the centrifuge tube and at the speed of 5000 rpm centrifugal 12 min, disposable medium, add 20 ml PBS and dissolved after ultrasonic broken blender 5 min, turn into the tube Centrifugal at the speed of 2000 rpm for 5 min to remove cell debris, and 15000 rpm centrifugal 12 min, disposable supernatant obtained precipitation for the bacterial extracts. Will be induced before and after induction of cell protein precipitation transferred to the same concentration for the entire electrophoretic analysis whether the large number of protein was expressed.(2): PTP-MEG2 Purification: Extract the Q-Sepharose Fast Flow ion-exchange column chromatography and SP-Sephadex ion-exchange column chromatography by the purified protein. Electrophoretic analysis purity above 95%.(3): PTP-MEG2 Characterization: Configurate different pH and ionic strength of the buffer, protein determination in different pH and ionic strength and temperature under the condition of enzymatic reaction. By its enzymatic reaction optimum conditions.3. PTP -MEG2 polyclonal antibody preparation and purificationWe use the purified PTP-MEG2 immune rabbit, sera obtained through PVDF fixed antigen affinity chromatography column and purified from the anti PTP-MEG2 polyclonal antibody. The ECL detection, purified antibody titer after 1:10000 can be reached (V/V); the sensitivite of antigen-antibody is 1 ng. The purified antibody titer can be reached 1:2500 (V/V); sensitivity antigen-antibody is 0.1 ng. Thus, the purified antibody, antigen sensitivite increased by 10 times; through the entire electrophoresis and Weston Blot Detection of antibody purity of more than 90% can be achieved, it is in the line with experimental immunization requirements. Purified antibody serum after 56℃, 30 min after heating inactivated by adding final concentration of 1/1000 of sodium azide, cryopreservation in -80℃.