PPF1基因C端片段在大肠杆菌中的融合表达和纯化以及抗体制备

PPF1是与G2豌豆短日不衰老现象紧密相关的基因之一 .以pSK PPF1为模板 ,用PCR方法得到了编码该蛋白C端的基因片段 ,称为PPFC .进一步将该基因片段克隆到中间载体pGEM T easy ,再酶切得到PPFC片段插入到pGEX 4T 1载体中 ,构建成表达质粒pGEX 4T PPFC .在BL2 1细胞中经IPTG诱导表达 ,得到GST PPFC融合蛋白 .用GST亲和柱纯化得到PPFC蛋白 ,将其作为抗原得到兔源的多克隆抗体 .Western杂交分析表明所得到的抗体具有较强的特异性 ,ELISA分析证实其效价为 10 6.该抗体的获得为用免疫沉淀等免疫分析技术研究PPF1与其它蛋白质的相互作用 ,从而阐明PPF1在延缓G2豌豆衰老中的分子机制提供了可能

Expression and Purification of PPF1 C-terminal Fusion Protein in E. coli as well as Antibody Preparation

PPF1 is one of the senescence-related genes cloned from G2 pea previously. The C terminal segment of this gene, named PPFC, was amplified by PCR from the original cloning vector, pSK-PPF1, and was further cloned into the T-easy vector to produce the new construct, pGEM-T-PPFC. The cloned PPFC segment was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector, pGEX-4T-1 to produce the expression vector pGEX-4T-PPFC. The recombinant plasmid was transformed into E. coli BL21. GST-PPFC fusion protein was obtained after the addition of IPTG into the growth media. This protein was purified by the GST affinity column and was injected into rabbit to produce polyclonal antibodies. Western blot analyses showed that the antibody reacted specifically in vitro to produced PPFC protein. ELISA analysis showed that the titer for this antibody is 10 6. This antibody made it possible to analyze the interaction between PPF1 and other proteins with immunoprecipitation or other immunological methods, and to elucidate the molecular mechanism of delaying G2 pea senescence by PPF1 protein.