粘着斑激酶的原核表达、纯化及抗体的制备

粘着斑激酶(focal adhesion kinase,FAK)是细胞质内单亚基非受体型酪氨酸激酶,通过各种信号途径参与调节细胞生长、发育、黏附、细胞骨架重组、转化、扩散和迁移等过程.采用PCR方法,从Flag-FAK质粒中克隆编码FAK C端273个氨基酸的基因片段,构建FAK融合蛋白原核表达载体pET28a( )/FAK,进行原核表达与蛋白纯化,取纯化的FAK蛋白免疫小鼠,制备FAK抗血清.结果表明构建的表达FAK C端功能结构域的原核表达质粒pET28a( )/FAK,经过BL21(DE3)大肠杆菌表达、镍亲和层析柱纯化,获得相对分子质量约33 kDa的融合蛋白,并利用小鼠制备了多克隆抗体,EL ISA检测显示该抗体有较高效价.荧光免疫结果显示此多克隆抗体与FAK蛋白特异性结合,为进一步研究神经细胞中FAK的作用机制奠定了基础.

Prokaryotic expression purification and polyclonal antibody preparation of focal adhesion kinase

Focal adhesion kinase(FAK) is a monosubunit inreceptor PTK,which participates to regulate cell growth,development,sticky,cytoskeleton recombination,inversion,diffusion and migration through signal transduction-pathway.The fragment of FAK gene encoding 273 amino acids in the C terminus was amplified from Flag-FAK plasmid by PCR.This fragment was then inserted into the prokaryotic expression vector to construct the recombinant plasmid pET28a( )/FAK,and expressed in Ecoli.BL21(DE3).After induction with IPTG,a molecular weight of 33 000 fusion protein was obtained and purified by Ni-NTA affinity chromatography.Mice were immunized with the purified FAK protein and the antiserum was obtained.The results of EL ISA and immunofluorescence indicated that the polyclonal antibody was of high titration and specificity,and can be used in the further research on FAK mechanism in nerve cell.