香蕉束顶病毒海口分离物核穿梭蛋白(NSP)的纯化及抗血清制备

NSP Protein Purification and Antiserum Preparation of Banana Bunchy Top Virus Isolate from Haikou

本研究以本实验室保存的含重组载体pDEST17-NSP的原核表达菌株E.coli BL21(DE3)为材料,于25℃、0.1mmol/L IPTG条件下诱导4h,集菌后超声波破碎,获得以包涵体形式表达的约20kD的融合蛋白。实验结果表明,将沉淀的融合蛋白溶于含6mol/L尿素的Binding Buffer中,再经Ni2+-NTA亲和层析纯化后,可获得高纯度的融合蛋白。将纯化融合蛋白经12%SDS-PAGE电泳,切胶回收目的带,液氮研磨并按1:1(W/V)混合佐剂,4次免疫家兔,获得BBTV病毒核穿梭蛋白的特异性抗血清。以融合蛋白作抗原,间接ELISA法测定其抗血清效价为1:5000。田间检测样品的最佳抗血清工作浓度为1:500。Western Blot鉴定结果表明抗血清能与目的蛋白特异性结合。本研究的结果将为下一步NSP基因转录调控和蛋白功能研究奠定一定基础。

Prokaryotic expression strain E. coli BL21 （DE3） of the recombinant vector pDEST 17-NSP was stored in our laboratory, and was induced at 20℃ with 0.1 mmol/L IPTG for 4 hours. Fusion protein of approximately 20 kD was expressed steadily as an inclusion body. The results showed that precipitation of fused protein was dissolved in Binding Buffer containing 6 mol/L urea, then fused protein solution was obtained by purifing of Ni^2＋-NTA agarose affinity chromatography. The purified fusion protein was extracted by 12% SDS-PAGE, and was triturated with liquid nitrogen, finally was mixed with adjuvant of 1：1 （W/V）. The rabbits was immunized for 4 times, and the specific antiserum of NSP of BBTV was acquired from the immunized rabbits later. The titer of the antiserum was higher than 1：5 000 by indirect enzyme-linked immunosorbent assay （ID-ELISA）, and the best work of concentration of 1：500 was determined in field testing. Western Blot analysis revealed that antiserum had the capability of specifical binding with the target protein. These investigations work should be establish basis for the research of transcription regulation ofNSP gene and its protein function.