【作者】 孙立文；

【导师】 付学奇；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2006， 硕士

【摘要】 为了研究人的适配子蛋白3BP2(又称作SH3BP2)在细胞信号转导中的生物学功能,本研究以3BP2的全长cDNA为模板,克隆构建了原核融合蛋白表达载体pGex2T-3BP2质粒,转化大肠杆菌DE3-plysS,进行融合蛋白质GST-3BP2的高效表达,并利用Gluthione交联的Sephorose-4B亲和层析柱纯化该融合蛋白质,制备了GST-3BP2。GST-3BP2免疫兔得到这个融合蛋白抗血清,通过抗体的负选择和正选择亲和层析柱进行纯化,制备了抗3BP2兔的多克隆抗体。利用免疫沉淀(Immunoprecipitation)和Western blot等生物学研究手段,在鼠脾中检测到了3BP2与SHP1的相互作用。3BP2作为酪氨酸磷酸激酶(PTK)Lyn,Syk的底物参与到与酪氨酸磷酸酶(PTP)SHP-1的信号通路中,这对于阐明与3BP2相关的TCR,BCR和FcεRI介导的信号通路,深入研究PTP和PTK之间的关系,为3BP2相关疾病过敏性疾病和巨颌症药物靶点的研究奠定了理论基础。更多还原

【Abstract】 In the quantities of regulation mechanisms of cell signaling transduction, thephosphorylation and dephosphorylation of proteins are of importance. Especially,phosphorylation of specific cellular proteins on tyrosine residues is one of the mostfundamental regulatory steps which dictates cell-cell communication, cell growthand proliferation, regulation of cell cycle and differentiation, tumorogenesis andtumor metastasis, nerve transmission, angiogenesis, embryogeny, and kinds ofhereditary and non-hereditary diseases. The research of protein tyrosinephophorylation has been hot spot in the field of biological science.The adaptor proteins which consist of several protein-protein binding motifoften act as the messenger between the activated cell-membrane acceptors andcytosolic signaling molecules. 3BP2 is an adaptor protein, which is 561 aa transcript consisting of threedomains: PH domain, Proline-rich region and SH2 domain, three tyrosinephophorylation sites 174,183,446 as well. As the bridge between activated cellmembrane receptors and various of intracellular cell signaling molecules, 3BP2exerts important roles on the multi-cellular functions, such as proliferation,transcription regulation, cell skeleton integration by the tyrosine phosphorylation.The relationship between PTPs and PTKs can be explored by implicating themechanism of 3BP2.For researching biophysical activities of the adaptor protein in the tissues andcells by immuno-precipitation, western blot and ELISA, the quantities of highspecific antibodies be needed. So the polyclonal antibody against 3BP2 is firstlyprepared. The detailed experimental steps as below: 1 The cloning and expression of 3BP2 geneThe 3BP2 was cloned and constructed into soluble fusion protein expressionvector pGex2T-HisC4, which was then sequenced and verified. Induced into E. coliDE3-LysS, the GST-3BP2 was highly expressed.There are proteins which molecular weight are almost 70kDa and 50kDa inSDS-PAGE, but not theorically 91kDa. The reason for uncomplete expression: (1)The cDNA sequence was verified;(2) The protein expression is same in other hostcells, such as E.coli DE3 Roseta and BL21;(3) Resulting from GST expression, Theinduced proteins was purified by GST affinity chromatography which imply noreading frame shift;(4) The qualities of induced proteins was 15-20% or so whichsuggested that is from the transformed expression construct.The polyclonal antibody is mainly from the antigen isotopes with amino acidsequence segments. Then the uncomplete protein expression should be suitable forantibody preparation.2 Isolation, purification of GST-3BP2The GST-3BP2 was isolated using GST affinity chromatography, which puritywas over 90% through the measurement of SDS-PAGE.3 The preparation of polyclonal antibody against 3BP2The antibody against 3BP2 was prepared and purified using negative selectivechromatography and PVDF immobilized antigen affinity (positive selective)chromatography, which is from rabbit immunized by GST-3BP2. The titers andantigen sensitivities examination was undergoned by ECL, which met the standardfor further experiments, then the 3BP2 in mouse spleen was identified by which theprepared antibody was verified.4 exploring the relationship between 3BP2 and SHP1Using immunoprecipitation and western blotting methods, The relationshipbetween 3BP2 and SHP1 was explored which interaction may influence on theinteraction between 3BP2 and PTKs, such as Lyn and Syk, furtherly TCR, BCR,FcεRI-mediated cell signaling pathways. The research will sound the base to revealthe relationship between PTP and PTK and drug target verification related to allergicdisease, cherubism.更多还原