本氏烟NbGAD1蛋白的原核表达及多克隆抗体制备

Prokaryotic expression and preparation of polyclonal antibody of NbGAD1 protein from Nicotiana benthamiana

γ-氨基丁酸(GABA)作为一种非蛋白质氨基酸,普遍存在于植物体内,在植物生长发育和抗胁迫方面发挥着重要作用。谷氨酸脱羧酶(GAD)是合成GABA的关键蛋白酶,目前关于GAD介导GABA调控植物防御病毒侵染的分子机制尚不清楚。利用RT-PCR克隆本氏烟NbGAD1基因,并与原核表达载体pET-28a连接,构建重组载体pET-28a-NbGAD1,诱导产生重组蛋白,经Ni-NTA柱纯化蛋白后免疫新西兰白兔,最终获得抗血清。结果表明:成功构建原核表达载体pET-28a-NbGAD1,经大肠埃希菌诱导纯化后获得分子量约57 ku的重组蛋白NbGAD1。免疫制备的抗血清经Western blot检测显示,抗血清效价达1∶100000,抗血清稀释2000倍仍能检测到浓度为0.005 mg·mL^(-1)的重组蛋白,且能检测到本氏烟中内源表达的NbGAD1蛋白,说明该抗血清特异性强、灵敏度高,为今后深入探究GAD在植物病毒侵染过程中的作用机制奠定了基础。

γ-aminobutyric acid is a non-protein amino acid,which is ubiquitous in plants,and GABA plays an important role in plant growth,development and stress resistance.Glutamate decarboxylase is a key protease for the synthesis of GABA.At present,the molecular mechanism of GAD-mediated GABA regulation against virus infection is unclear in plants.The NbGAD1 gene of Nicotiana benthamiana was cloned by RT-PCR,and connected with the prokaryotic expression vector pET-28a to construct the recombinant vector pET-28a-NbGAD1.The recombinant protein was induced and purified by Ni-NTA column,and the antiserum was obtained by immunizing New Zealand White rabbits.The results showed that the prokaryotic expression vector pET-28a-NbGAD1 was successfully constructed,and the recombinant protein NbGAD1 with molecular weight of about 57 ku was obtained after induction and purification from Escherichia coli.Western blotting results showed that the titer of the antiserum reached 1∶100000,and the recombinant protein with the density of 0.005 mg·mL^(-1)could still be detected by the antiserum diluted 2000 times.In addition,the endogenous NbGAD1 protein from N.benthamiana can be also detected using the antiserum.Overall,the antiserum has the advantages of strong specificity and high sensitivity,which provides a basis for further study on the mechanism of GAD in plant virus infection.