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目的以原核表达的人高迁移率族B1(Highmobilitygroupbox1,HMGB1)蛋白为免疫原免疫日本大耳白兔,制备其兔多克隆抗体并进行鉴定。方法将人HMGB1cDNA克隆于原核表达载体pQE-80L并转化大肠杆菌DH5α,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导HMGB1表达,Ni2+-NTA层析纯化后,用其免疫兔。以ELISA检测抗体效价,Westernblot鉴定抗体特异性。结果成功表达并纯化了人HMGB1蛋白,纯化后纯度可达96%;ELISA法测定抗体效价为1∶25600;Westernblot结果显示所制备的抗体可特异性与人HMGB1蛋白结合。结论成功制备了兔抗人HMGB1多克隆抗体,为进一步研究HMGB1与相关疾病的关系打下了基础。
OBJECTIVE: To immunize Japanese white rabbits with prokaryotic human high mobility group box 1 (HMGB1) protein B1 to prepare and identify their polyclonal antibodies. Methods Human HMGB1 cDNA was cloned into prokaryotic expression vector pQE-80L and transformed into E. coli DH5α. HMGB1 expression was induced by isopropyl-β-D-thiogalactoside (IPTG). After purification by Ni2 + -NTA chromatography, rabbit. Antibody titers were detected by ELISA and Western blot to identify antibody specificity. Results The human HMGB1 protein was successfully expressed and purified. The purity of the purified protein reached 96% after purification. The antibody titer was 1:25600 by ELISA. The result of Western blot showed that the antibody could specifically bind to human HMGB1 protein. Conclusion The rabbit anti-human HMGB1 polyclonal antibody was successfully prepared, which laid the foundation for further study on the relationship between HMGB1 and related diseases.