ORP4L多克隆抗体的制备及其在蛋白质组学研究中的应用水

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ORP4L382-485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 sepharosC 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—ernblotting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 eDNA 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 purified, New Zealand rabbits were immunized by using the purified GST- ORP4Lm protein, polyelonal antibody against ORP4L was obtained and further confirmed by Western blotting. The antibody was then conjugated to sepharose beads and the cell lysis of stable expression ORP4L was flowed through the beads. The potential interacting proteins with ORP4L were collected and analyzed by mass spectrometry. 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 p01yclonal antibody with high specificity has been prepared, and it could be applied for ORP4L proteomics resolution.