（1. 南京师范大学海洋科学与工程学院，南京 210023；2. 江苏省农业科学院兽医研究所，农业农村部兽用生物制品工程重点实验室，南京 210014；3. 南京农业大学生命科学院，南京 210095；4. 兽用生物制品（泰州）国泰技术创新中心，泰州 225300）
摘　要：铁蛋白是普遍存在于各类有核生物、参与机体铁离子稳态调控的一类蛋白质，它可以自组装形成14 nm左右的中空笼状颗粒，常被作为药物和疫苗的递送载体。支原体铁蛋白的晶体结构显示，铁氧化活性中心和铁离子通道与其他物种存在结构上的差异。目前螺原体铁蛋白还未有相关研究。本研究发现，螺原体铁蛋白与包含支原体在内的其他物种铁蛋白均具有较低的同源性。利用大肠杆菌表达中华绒螯蟹螺原体铁蛋白（SeFer），经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，蛋白能以可溶的形式表达于大肠杆菌细胞内。分别通过二乙氨基乙基（DEAE）弱阴离子交换层析、分子筛层析，获得高纯度的SeFer。在4℃和18℃，用坐滴气相扩散法对SeFer进行晶体筛选，该蛋白在多种条件下能生长晶体，通过同步辐射光源衍射，其晶体最好衍射至2.90 Å，属于I432空间群，收集的数据可用于后继结构解析。非变性聚丙烯酰胺凝胶电泳（Native-PAGE）、动态光散射（DLS）以及SeFer分子筛出峰位置显示，经大肠杆菌重组表达的SeFer相对分子质量为400 kD左右，粒径大小为15 nm左右，与理论值接近。利用Alphafold 2对SeFer进行结构预测，发现其具有与其他铁蛋白类似的二十面体球状结构，但是其铁氧化活性中心与支原体铁蛋白保守位点存在差异。本结果不仅为研究SeFer的晶体结构提供了基础，也为疫苗载体提供了新的候选。
关键词：中华绒螯蟹螺原体；铁蛋白；蛋白纯化；晶体结构
中图分类号：Q71                                 文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.03.005

 (1. School of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China; 2. Key Laboratory of Veterinary Biological Products Engineering, Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 3. School of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China; 4. Guotai Technology Innovation Center of Veterinary Biological Products (Taizhou), Taizhou 225300, China)
Abstract: Ferritin is a class of proteins ubiquitously found in various types of nucleated organisms and involved in the iron homeostasis regulation of body. Ferritin can self-assemble to form a hollow nanocage of about 14 nm in size and is often used as carrier for drug delivery and vaccine construction. The crystal structure of Mycoplasma ferritin shows atypical ferroxidase centres and new pathway for Fe2+ uptake, which are structurally different from those of other species. Not much work has been reported for Spiroplasma ferritin. Here, our studies show that Spiroplasma ferritin has low sequence similarity with other ferritins including Mycoplasma ferritins. Spiroplasma eriocheiris Ferritin (SeFer) was recombinant expressed in E. coli in soluble form after IPTG (isopropyl-beta-D-thiogalactopyranoside) induction. High-purity SeFer protein was obtained after purification by DEAE (diethylaminoethyl) weak anion exchange chromatography and gel filtration. Crystal screen of SeFer was performed by sitting drop vapor diffusion at 4℃ and 18℃. SeFer crystals grew in several screen conditions. The best crystal diffracted to 2.90 Å by synchrotron radiation source and it belonged to the I432 space group. The X-ray crystallography data collected here can be used for further structure determination. Native-PAGE, dynamic light scattering, and the position of peaks of SeFer in molecular sieve showed that the molecular weight of SeFer is about 400.0 kD, and the diameter of SeFer was about 15 nm, both of them are close to the theoretical value. The structural model of SeFer was predicted by using Alphafold 2. The predicted model shows that SeFer has a regular icosahedral overall structure, which is similar to other ferritins. However, SeFer model shows that the key residues of ferroxidase center differ from those of mycoplasma ferritin. Our results not only provide a basis for studying the crystal structure of SeFer, but also provide new candidates for vaccine vectors.
Key words: Spiroplasma eriocheiris; ferritin; protein purification; crystal structure
（Acta Laser Biology Sinica, 2024, 33(3): 227-235）