ORP4L 多克隆抗体的制备及其在蛋白质组学研究中的应用

目的:原核表达并纯化人氧化固醇结合蛋白相关蛋白4(ORP4L)肽段,制备兔抗人ORP4L多克隆抗体,并利用其进行蛋白质组学研究。方法:应用PCR技术扩增人ORP4L 382-485氨基酸(ORP4Lm)的基因序列并插入到PGEX-4T-1载体中,在大肠杆菌RosettaTM(DE3)中表达融合蛋白GST-ORP4Lm。利用所表达的融合蛋白中含有的GST标签进行亲和纯化。用所获得的纯化蛋白免疫新西兰大白兔,获得兔抗人ORP4L多克隆抗体。用Western blotting检测抗体免疫特异性。将亲和纯化后的抗体偶联到CNBr-actived sepharose beads上,利用免疫沉淀的方法,通过质谱仪分析鉴定可能与ORP4L存在相互作用的蛋白质。通过West-ern blotting进一步确证特异性的相互作用蛋白。结果:在大肠杆菌中表达并纯化了GST-ORP4Lm重组蛋白,用其免疫新西兰大兔,成功制备了相应兔源多克隆抗体,Western blotting证实该抗体可以特异识别内源性及外源性的ORP4L蛋白。质谱分析和Western blotting的结果表明所制备的多克隆抗体可以用于蛋白质组学研究。结论:利用重组的GST-ORP4Lm融合蛋白成功制备了有良好特异性的ORP4L多克隆抗体,并可将其用于ORP4L的蛋白组学研究。

Preparation and Application in Proteomics Approach of Polyclonal Antibody against Human ORP4L

Objective: To prepare polyclonal ORP4L antibody and utilize it for ORP4L proteomics resolution.Methods: The cDNA of ORP4L 382-485 amino acids residues(ORP4Lm) amplified by the polymerase chain reaction(PCR) was inserted into the PGEX-4T-1 vector.The plasmid was transformed into RosettaTM(DE3) and the recombinant fusion protein GST-ORP4Lm was expressed and puri-fied,New Zealand rabbits were immunized by using the purified GST-ORP4Lm protein,polyclonal antibody against ORP4L was ob-tained and further confirmed by Western blotting.The antibody was then conjugated to sepharose beads and the cell lysis of stable expres-sion ORP4L was flowed through the beads.The potential interacting proteins with ORP4L were collected and analyzed by mass spec-trometry.The result of the mass spectrometry was assessed by Western blotting.Results: The rabbit anti-ORP4L antibody was prepared.The antibody was able to detect both endogenous and exogenous ORP4L.ORP4L proteomics was carried out successfully.Conclusion: The rabbit anti-ORP4L polyclonal antibody with high specificity has been prepared,and it could be applied for ORP4L proteomics reso-lution.