葡萄β型液泡加工酶基因(VvβVPE)的原核表达、纯化与多克隆抗体制备

摘要

液泡加工酶(VPE)基因是植物内启动和执行细胞程序性死亡的关键基因,β型VPE是种子特异表达并且为种子蛋白成熟所必须的基因.本研究以黑比诺葡萄(Vitis vinifera cv.Pinot Noir)花后8个不同时期胚珠的cDNA混合样为模板,通过RT-PCR扩增得到葡萄β型液泡加工酶基因(暂命名VvβVPE,GenBank登录号:KC136352),开放阅读框1 485 bp.进一步将VvβVPE克隆到pGEX-6P-1原核表达载体并转入大肠杆菌(Escherichia coli)BL21中,经IPTG诱导,获得约81.3 kD的包涵体融合蛋白GST-VvβVPE.将诱导条件优化后,在37℃、0.05 mmol/L IPTG诱导5h后融合蛋白GST-VvβVPE的表达量最大,采用电透析与盐酸胍处理后透析复性两种方法获得纯化包涵体蛋白,电透析纯化法获得纯化包涵体蛋白的浓度可达到免疫要求.使用纯化蛋白免疫新西兰大白兔制备多克隆抗血清,经Western blot检测,该抗血清(稀释到1∶10 000)与GST-VvβVPE融合蛋白识别反应良好,获得的抗血清可用于以后的VvβVPE的酶活性分析、免疫亚细胞定位分析以及转基因植株蛋白水平的鉴定等,为进一步明确VvβVPE在葡萄胚珠发育中的作用提供基础数据.%Vacuole processing enzyme(VPE) gene family plays an important role in mediating and executing programmed cell death in plants.β type VPE is specifically expressed in seed,which is indispensable in seed development.The ovules were harvested from V.vinifera cv.Pinot Noir in 8 different development stages.RNA was isolated from these ovules.VvβVPE gene was obtained by RT-PCR,and the mixture of cDNA was used as template.The results showed VvβVPE gene contained a complete ORF(open reading frame) sequence (1 485 bp) encoding 494 amino acid(GenBank accession number:KC136352).The full-length sequence was cloned into the prokaryotic expression vector of pGEX-6P-1.An about 81.3 kD fusion protein of inclusion body (GST-VvβVPE) was expressed in the Escherichia coli BL21 through IPTG inducing.After optimizing the inducing condition,two different methods were adopted to purify the protein,containing electro dialysis and dialysis renaturation by GdmC1 treating,and then immunized the rabbit to obtain the polyclonal antibody.Western blot displayed a specific immunity-antigen signal,suggesting the obtained antiserum can be used for function analysis and detecting transgenic plants.