The Cloning And Expression Of Human SHP-1 Catalytic Domain And The Preparation Of Its Polyclonal Antibody

In the quantities of regulation mechanisms of cell signaltransduction, the phosphorylation and dephosphorylation of proteinsare of importance. Especially, phosphorylation of specific cellularproteins on tyrosyl residues is one of the most fundamental regulatorysteps which dictates cell-cell communication, cell growth andproliferation, regulation of cell cycle and differentiation,tumorogenesis and tumor metastasis, nerve transmission, angiogenesis,embryogeny, and kinds of hereditary and non-hereditary diseases. Theresearch of protein tyrosine phophorylation has been hot spot in thefield of biological science.SHP-1 (also called HCP, SHPTP1 or PTP1C), SH2-containing tyrosinephosphatase-1, is a kind of cytoplasm protein. On the N-terminal ofSHP-1 there are two SH2 domains, and on the C-terminal there is onedomain with the function of catalysis. The interaction of SHP-1 andITIM which is near to membrane is key for conducting inhibitors.SHP-1 can catalyze the dephosphorylation of Tyr so as todown-regulate the signal transduction of many receptors, and can exertthe same influence on the activation of NK cells and ADCC .Protein tyrosine phosphatase SHP-1 may regulate the functions ofB cell, T cell and NK cell. In many tumor ,leukemia cell lines andmany clinical tumor specimens, it can be detected that the expressionof SHP-1 is blocked or decreases distinctly. Therefore, SHP-1 has thefunction of inhibiting the tumor development. Through SHP-1interacting with the cell factor receptor to dephosphorylate JAK, bloodcells of the mice with SHP-1 domain defective are abnormal whichindicates that cytoplasm tyrosine phosphatase is related to thedifferentiation of blood cells. SHP-1 is one of important PTP subfamily with SH2domain. Forresearching biophysical activities of the enzyme in the tissues and cellsby immuno-precipitation, western blot and ELISA, the quantities ofhigh specific antibodies be needed. Meanwhile, process of antibodypreparation and puricification is explored to construct the experimentplatform of my laboratory. The catalytic domain of △SHP-1 cDNA is cloned ,constructedinto the vector pT7 with Amp resistance, then expressed in the E.coliBL21. After isolating and purifying by ion chromatography, the enzymecharacterization of is undergone. rabbit serum after immunized by△SHP-1 is purified so as to produce high specific polyclonalantibody.1. The Cloning and Expression of Intracellular Domain of SHP-1cDNA SHP-1 cDNA as template,the catalytic domain of which,i.e. ΔSHP-1,is amplified by PCR, in which the NdeI and HindIII restrictionenzyme digestion site is conducted at the N terminal and C terminal .ΔSHP-1 in the PCR products investigated by agar electrophoresis isligated into the PKS clone vector which is digested by EcoRI. Afterthat, PKS-ΔSHP-1 is digested by Nde I and Hind III ,check out thenconstructed into pT7 expression vector. pT7-ΔSHP-1 is delt with T4DNA ligase, determined by enzyme digestion map,then transformedinto E.coli BL21,which is highly expressed.0000000000000000002. Isolation, Purification and enzymecharacterization of Intracellular Domain of SHP-1 After isolation and purification by ion chromatography,Q-Sepharose Fast Flow and SP-Sephadex, the interested protein ΔSHP-1 is dialyzed, lyophilized, then subjected to SDS-PAGE andHPLC ,the purity of which is over 95%. The study of the enzyme characterization indicates that theoptimal pH, the optimal reaction temperature , and the optimal ionintensity of ΔSHP-1 is 5.0, 36oC and 0 respectively when p-NPP assubstrate; by research of enzyme kinetics, the kinetics parameters Kmand Vmax is 22.0mM and 2. 901μmol/min respectively.3. The Preparation and Purification of Polyclonal Antibody of ΔSHP-1 The rabbit was immunized by antigen of purified ΔSHP-1. Theantibody against SHP-1 was prepared and purified from serum byPVDF immobilized antigen affinity chromatography. Before and afterpurification of antibody, by means of ECL , the both efficency were1:10000, the sensitivity is 0.01μg and 1 ng respectively which isincreased to 10 fold. The purity of antibody is over 90% by theinvestigation of SDS-GAGE and western blot. These results indicatedthe antibody match to and surpass the standard for further experiments.