EB病毒GST-Rta融合蛋白的表达、纯化及其多克隆抗体的制备

Expression and Purification of GST-Rta Fusion Protein from EB Virus and Preparation of the Polyclonal Antibody Against GST-Rta

目的表达和纯化EB病毒的重组融合蛋白谷胱氨肽S转移酶(GST)-Rta185和GST-Rta150,并制备特异性多克隆抗体。方法质粒表达载体pGEX-R185I、pGEX-R150I和pGEX-5X-3分别转入大肠杆菌BL21(DE3)中,异丙基硫代-β-D半乳糖苷(IPTG)诱导表达,用GlutathioneSepharose4B亲和层析柱纯化目的蛋白。将纯化后的GST-R185、GST-R150和GST蛋白免疫新西兰大白兔制备抗血清。结果在细菌裂解液中检测到高表达量的融合蛋白,经GlutathioneSepharose4B亲和层析柱纯化获得较高纯度的GST-Rta融合蛋白。应用Westernblot和ELISA方法检测证实,用纯化的GST-Rta蛋白免疫家兔得到了特异性的多克隆抗体。结论通过上述方法制备出来的融合蛋白纯度较高,免疫家兔获得的多克隆抗体具有良好的特异性,为研究EB病毒Rta蛋白及与EB病毒相关疾病的诊断和治疗提供了重要的条件。

Objective To obtain the recombinant fusion proteins GST-Rta185 and GST-Rta150 from EB virus and prepare two Rta protein specific polyclonal antibodies, respectively. Methods Plasmids pGEX-R150I, pGEX-R185I and pGEX-5X-3 were separately transformed into Escherichia coli BL21 （DE3）. Expressions of the recombinant proteins R150-GST, R185-GST and free GST were induced by 0.1mmol/L IPTG in LB medium. The expressed proteins were purified from lysates with Glutathione Sepharose 4B. Purified proteins were mixed with Freund＇s adjuvant and then were used to immunize rabbits. Results High levels of expression of target proteins were detected in the lysates and the purified proteins were obtained by affinity chromatography with Glutathione Sepharose 4B. Western blot and ELISA analysis suggested that the polyclonal antibodies against GST-R185 and GST-R150 were specific. Conclusion The antiserums have good specificity. They are important for the research on Rta fusion proteins from EB virus and for the diagnosis or treatment of EB virus associated diseases.