嗜水气单胞菌RecA和Gap-2作为内参蛋白的评价

Evaluation of RecA and Gap-2 in Aeromonas hydrophila as internal reference proteins

为确定嗜水气单胞菌中可用于Western Blot的适宜内参蛋白,考察了重组酶RecA和3-磷酸甘油醛脱氢酶Gap-2在该菌不同培养阶段及培养条件下的表达稳定性。以嗜水气单胞菌基因组为模板扩增recA和gap-2基因,并对蛋白进行异源表达。利用亲和层析和凝胶过滤层析技术纯化蛋白,制备多克隆抗体。通过Western Blot技术研究了RecA和Gap-2蛋白在嗜水气单胞菌不同培养时间、不同培养温度和培养环境中铁离子浓度差异条件下的表达情况。结果显示,重组表达载体pET-28a-recA和pET-28a-gap-2构建成功,可与His标签融合表达。纯化后得到了纯度较高的RecA和Gap-2,以二者为抗原制备的多克隆抗体效价均达到1∶512000。Western Blot结果表明,RecA在细菌生长稳定期表达情况与前期有所差异,而Gap-2蛋白在该菌不同培养时期、温度及铁离子浓度差异的环境中表达较为恒定。研究表明,Gap-2更适合作为嗜水气单胞菌Western Blot检测用内参蛋白。

To determine the suitable internal reference protein for Western Blot in Aeromonas hydrophila,the expression stability of recombinase RecA and glyceraldehyde 3-phosphate dehydrogenase Gap-2 in different culture stages and culture conditions were investigated.Using Aeromonas hydrophila genome as template,recA and gap-2 genes were amplified,and the protein was heterologously expressed.Affinity chromatography and gel filtration chromatography were used to purify proteins and prepare polyclonal antibodies.Western Blot was used to study the expression of RecA and Gap-2 proteins in Aeromonas hydrophilaat different culture time,culture temperature and iron ions concentration in culture environment.The results showed that the recombinant expression vectors pET-28a-recA and pET-28a-gap-2 were successfully constructed and could be fused with His tag.After purification,RecA and Gap-2 with high purity were obtained.The titers of polyclonal antibodies prepared with them as antigens reached 1∶512000.Western Blot result showed that the expression of RecA in the stable period of bacterial growth was different from that in the early stage,while the expression of Gap-2 protein was relatively constant in the environment with different culture periods,temperatures and iron ion concentrations.The results indicated that protein Gap-2 was more suitable as an internal reference protein for Western Blot detection of Aeromonas hydrophila.