The Cloning And Expression Of Human PTPMEG1 Catalytic Domain And The Preparation Of Its Polyclonal Antibody

In the quantities of regulation mechanisms of cell signal transduction, the phosphorylation and dephosphorylation of proteins are of importance. Especially, phosphorylation of specific cellular proteins on tyrosyl residues is one of the most fundamental regulatory steps which dictates cell-cell communication, cell growth and proliferation, regulation of cell cycle and differentiation, tumorogenesis and tumor metastasis, nerve transmission, angiogenesis, embryogeny, and kinds of hereditary and non-hereditary diseases. The research of protein tyrosine phophorylation has been hot spot in the field of biological science.Protein tyrosine phosphatase MEG1 is a member of a PTPase subfamily, this subfamily also contain PTPH1, PTPBAS, and PTPD1. PTPMEG1 contains several characteristic motifs outside the catalytic domain, including an aminoterminal 300 amino acid domain that is homologous to erythrocyte cytoskeleton protein 4.1 followed by two praline-rich sequences that have potential to bind to SH3 domain, two PEST sequences that predict rapid proteolysis, and a 100 amino acid motif designated DHR domain.Most of the members of the protein 4.1-domain- containing protein family have been immunolocalized to cell-cell adhesion junction, focal adhesion plaques, or cell-cell contact regions. DHR-domain-containing protein are also found mainly associated with cell-cell junctions. Together with its unique structure and the effects that have been observed. PTPMEG1 is likely to be involved in the organization of cell-cell contacts or in intercellularcommunication. PTPMEG1 interacts with glutamate receptor 82 and s subunits. And it may be concerted with osteoporosis and cancerometasis.PTPMEG1 is one of important FTP subfamily containing band protein 4.1 domain. For researching biophysical activities of the enzyme in the tissues and cells by immuno-precipitation, western blot and ELISA, the quantities of high specific antibodies be needed. Meanwhile, process of antibody preparation and puricification is explored to construct the experiment platform of my laboratory.The catalytic domain of PTPMEG1 cDNA is cloned .constructed into the vector pT7 with Amp resistance, then expressed in the E.coli Roseta DE3. After isolating and purifying by preparative electrophoresis. Rabbit serum after immunized by APTPMEG1 is purified so as to produce high specific polyclonal antibody.1. Clone and Expression the Catalytic Domain Gene of PTPMEG1 pBluescript IIKS-MEG1 as the template, amplified the catalytic domaingene of PTPMEG1 by PCR, in which the Nde I, EcoR I restriction enzyme digestion site and stop codon are conducted at the N terminal and C terminal. and constructed expression vector pT7-AMEGl that was cut by Ndel and EcoRl, and was identified by nucleic acid electrophoresis. Then we amplified it, transformed it into E. coli Roseta DE3 and obtained high expression strain.2. Segregation and Identification of APTPMEGlWe used supersonic quassation to handle the bacterias we had collected, then centrifuge them to collect the praecipitatums that were identified by protein electrophoresis. Purified the proteins by preparative electrophoresis, and then determined the number of EP tubes that contained our aimed proteins,and then concentrated the proteins in tubes that only contained our aimed proteins and determined the concentration of concentrated solution. 3. The Preparation and Purification of Polyclonal Antibody of APTP MEG1 The rabbit was immunized by antigen of purified PTPAMEGl. The antibody against APTP MEG1 was prepared and purified from serum by PVDF immobilized antigen affinity chromatography. Before and after purification of antibody, by means of ECL, the both efficency were 1:10000, the sensitivity is 0.1 ng and 1 ng respectively which is increased to 10 fold. The purity of antibody is over 90% by the investigation of SDS-GAGE and western blot. These results indicated the antibody match to and surpass the standard for further experiments. The purified antibody is dealt with at 56 °C for 30 min ,added by azide sodium with final concentration l/1000(W/V) then fractioned which was stored under -80 °C.All the work above provide us the basis for the study of physiological function of APTP MEG1 at the level of cell and its effect in cell signal transduction.