目的原核表达纯化幽门螺杆菌σ~(54)蛋白,并制备σ~(54)蛋白的多克隆抗体。方法以幽门螺杆菌26695基因组为模板扩增完整的rpo N基因片段,并将其克隆到含有His标签编码序列的原核表达载体p Tri ExTM-4上,将重组质粒转化大肠杆菌JM109DE感受态细胞,在IPTG诱导下表达带有His标签的融合蛋白,利用His-Bind亲和柱进行蛋白的纯化,将纯化的蛋白辅以佐剂免疫家兔,制备σ~(54)蛋白的多克隆抗体。结果σ~(54)蛋白在大肠杆菌JM109DE菌株中有较高表达,纯化后进行SDS-PAGE获得了与预期融合蛋白大小一致的单一条带。获得的σ~(54)蛋白多克隆抗体具有较好的σ~(54)蛋白结合性和特异性。结论成功表达和纯化了σ~(54)蛋白并制备了多克隆抗体。 Objective To purify the Helicobacter pylori (54) protein from prokaryotic cells and prepare the polyclonal antibody of σ ~ (54) protein. Methods The full-length rpo N gene fragment was amplified by using the Helicobacter pylori 26695 genome and cloned into the prokaryotic expression vector pTit ExTM-4 containing the His-tagged coding sequence. The recombinant plasmid was transformed into E. coli JM109DE competent cells, The fusion protein with His-tag was expressed under the induction of IPTG, the protein was purified by His-Bind affinity column, and the purified protein supplemented with adjuvant was used to immunize rabbits to prepare the polyclonal antibody of σ ~ (54) protein. Results The σ ~ (54) protein was highly expressed in Escherichia coli JM109DE strain. After purification, a single band consistent with the size of the expected fusion protein was obtained by SDS-PAGE. The obtained polyclonal antibody of σ ~ (54) protein has better binding and specificity of σ ~ (54) protein. Conclusion The σ 54 protein was successfully expressed and purified and the polyclonal antibody was prepared.