苜蓿中华根瘤菌烯酯酰ACP还原酶FABI2的原核表达纯化以及多克隆抗体的制备

Prokaryotic Expression and Purification of Allyl Acyl ACP Reductase in Sinorhizobium meliloti and Preparation of Polyclonal Antibody

烯酯酰ACP还原酶基因fab I是脂肪酸合成途径中的关键基因,苜蓿中华根瘤菌(Sinorhizobium meliloti)基因组中有这一基因的同源基因fab I1和fab I2,为了对中华苜蓿根瘤菌烯酯酰ACP还原酶基因fab I2的功能进行深入研究,本实验进行该基因多克隆抗体的制备.从已有的pET-28b-FABI2质粒中扩增出目的片段,构建了带有GST标签的原核表达载体p GEX-2T-FABI2,将两个标签的表达载体转入大肠杆菌(Escherichiacoli)BL21(DE3)后,在0.1,mmol/L IPTG、20,℃、185,r/min条件下诱导6,h,分别获得相对分子质量约为2.8×104、5.4×104表达fab I2的重组蛋白,且均在上清液中以可溶性蛋白的形式存在.将经Ni柱亲和纯化的6×His-FABI2融合蛋白免疫新西兰大白兔,4次免疫后测效价达256,000.进一步将抗血清经过ProteinA纯化获得了高特异性和灵敏度的多克隆抗体.以得到的抗体为一抗,另一个标签的抗原上样进行免疫印迹(Westernblot)检测,为单一条带,说明制备的多克隆抗体能够很好地识别FABI2蛋白,并且排除了标签所产生的抗体对Western blot结果的影响.

Allyl acyl ACP reductase gene fabI is the key gene in the synthesis of fatty acid,and fabI1 and fabI2 are its homologous genes in Sinorhizobium meliloti genome.In order to further study the function of the allyl acyl ACP reductase gene fabI in Sinorhizobium meliloti,polyclonal antibody was prepared.Target fragments were amplified out from pET-28b-FABI2 plasmid,and a GST-tagged prokaryotic expression vector of fabI2 gene from Sinorhizobium meliloti was constructed.The fusion protein was expressed in Escherichia coli BL21(DE3).Uunder the conditions of 0.1 mmol/L IPTG,20,℃,185 r/min and induced for 6 hours,the molecular weight of the fusion proteins was 2.8×104 and 5.4×104,and could exist in supernatant in the form of soluble protein.New Zealand white rabbits were immunized with the Ni-column affinity purified 6×His-FABI2 fusion protein.After four immunizations,their titer reached 256,000.The antiserum was further purified with Protein A to obtain highly specific and sensitive polyclonal antibodies.The prepared antibody was used as primary antibody,and GST-tagged expressed protein as a sample,the results of Western blot showed that the polyclonal anti-body was very good at recognizing the FABI2 protein in Sinorhizobium meliloti,and the effect of the antibodies produced by labels on the results of Western blot was excluded.