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目的表达并纯化βB2-晶体蛋白,制备其多克隆抗体,用以研究βB2-晶体蛋白聚合的机制。方法用RT-PCR的方法扩增了βB2-晶体蛋白的cDNA并克隆到原核表达载体pMON5473中。用萘啶酮酸诱导recA启动子后,蛋白质在大肠杆菌中得到了高效表达。表达的蛋白质用DEAE纤维素层析和凝胶过滤纯化,SDS-PAGE显示一条带。与佐剂混合后免疫新西兰大白兔。结果纯化了大鼠βB2-晶体蛋白,成功制备了其多克隆抗体。结论制备的多克隆抗体为下阶段的研究提供了重要的实验材料。
Objective To express and purify βB2-crystal protein and prepare its polyclonal antibody to study the mechanism of βB2-crystal protein polymerization. Methods The cDNA of βB2-crystal protein was amplified by RT-PCR and cloned into prokaryotic expression vector pMON5473. After the recA promoter was induced by nalidixic acid, the protein was highly expressed in E. coli. The expressed protein was purified by DEAE cellulose chromatography and gel filtration, and one band was shown by SDS-PAGE. New Zealand white rabbits were immunized with adjuvant. Results The rat βB2-crystallin was purified and its polyclonal antibody was successfully prepared. Conclusion The prepared polyclonal antibody provides important experimental materials for the next stage of research.