金黄葡萄球菌蛋白A与SUMO标签的融合表达及应用研究

目的 从金黄葡萄球菌菌株(ATCC6538)中克隆得到蛋白A的全长基因并对其结构及应用进行研究.方法 从该菌株的基因组DNA中克隆得到全长的葡萄球菌蛋白A基因,对其基因结构分析之后,将该基因的成熟区全长片段和抗体结合功能区片段重组到pHisSUMO原核分泌表达载体上与SUMO标签融合表达,通过ELISA的方法来鉴定融合蛋白的抗体结合活性及其稳定性,并将抗体结合功能区片段耦联到CNBr活化的琼脂糖凝胶上进行抗体的纯化.结果 首次获得了此株菌的全长蛋白A基因并发布于GenBank中,登录号为EU695225.蛋白A全长蛋白和功能区蛋白在pHisSUMO载体上得到正确高效表达,通过ELISA鉴定SUMO标签的存在,没有影响全长蛋白和功能区蛋白的抗体结合活性并有效提高了功能区蛋白的稳定性.在抗体纯化实验中,应用表达的蛋白A能够得到浓度和纯度较高的抗体并具有较好的效价.结论 克隆得到一个新的葡萄球菌蛋白A全长基因.蛋白A功能区蛋白与SUMO标签融合表达,在保持抗体结合活性的基础上提高了稳定性,并成功应用于抗体的纯化.

Expression, purification and application of staphylococcal protein A fused to SUMO

Objective To clone the full length staphylococcal protein A(SPA) gene from Staphylococcus aureus (ATCC6538), and subsequently study the gene structure and antibody binding ability.Methods The full length and the functional region of the SPA gene were cloned into pHisSUMO vector respectively, and expressed in E. coli. The full length and the functional fragment of the SPA protein were detected for antibody binding ability and stability. The functional fragment of the SPA protein fused with SUMO was coupled to the CNBr-activated agarose for antibody purification from rabbit serum. Results A variant of the full length SPA gene was cloned, which has been submitted to GenBank (the accession number is EU695225). Two fusion proteins had the same antibody binding ability as the untagged SPA protein. However, the formers was more stable than the latter at the tested conditions. SUMO-SPA conjugated-agarose kept high efficiency for antibody binding. Conclusion To our knowledge, the full length SPA gene of S.aureus(ATCC6538) is a novel variant. The SUMO tag can improve the stability of the functional region of the SPA protein without damaging the antibody binding ability. This fusion protein has been used for antibody purification successfully.