Page 37 - 南京医科大学学报自然科学版

第43卷第2期 南京医科大学学报（自然科学版）
2023年2月 Journal of Nanjing Medical University（Natural Sciences） ·179 ·

·基础医学·

小鼠 IL⁃3 的原核表达条件优化及其促肥大细胞分化成熟的活

性研究

胡昊扬 ，许志强 2
1
南通大学医学院，江苏 南通 226019；南京医科大学第一附属医院药学部，江苏 南京 210029
1 2

［摘 要］ 目的：对小鼠白细胞介素3（interleukin⁃3，IL⁃3）进行原核表达条件筛选和优化，为体外肥大细胞模型的高效率构建
奠定物质基础。方法：根据小鼠IL⁃3的核苷酸序列进行大肠杆菌偏爱密码子优化并构建至pET⁃28a（+）质粒中，随后转化至大
肠杆菌BL21（DE3）并筛选小鼠IL⁃3在该体系下表达的最佳条件，结合镍亲和层析和离子交换层析对其进行纯化，验证其促进
小鼠骨髓细胞分化为肥大细胞的活性。结果：成功构建pET⁃28a（+）⁃IL⁃3原核表达载体，诱导出在SDS⁃PAGE中显示约16 kDa
的蛋白条带，并通过四因素多水平参数筛选确定了最佳的诱导条件。最终纯化的小鼠IL⁃3与干细胞生长因子联合诱导小鼠骨
髓细胞分化可成功获得表面FcεRI和CD117双阳性的肥大细胞，且具有β⁃己糖胺酶释放功能。结论：构建了小鼠IL⁃3的表达
载体并筛选了最佳的原核表达条件，所纯化的小鼠IL⁃3能够成功用于过敏反应研究的体外肥大细胞模型构建。
［关键词］ 白细胞介素3；肥大细胞；大肠杆菌；原核表达
［中图分类号］ R329.26 ［文献标志码］ A ［文章编号］ 1007⁃4368（2023）02⁃179⁃08
doi：10.7655/NYDXBNS20230205

Optimization of the prokaryotic expression of mouse interleukin⁃3 and investigation of its
bioactivity in mast cell differentiation

1 2
HU Haoyang ，XU Zhiqiang
2
1 School of Medicine，Nantong University，Nantong 226019；Department of Pharmacy，the First Affiliated Hospital of
Nanjing Medical University，Nanjing 210029，China

［Abstract］ Objective：In this study，the conditions for the prokaryotic expression of mouse interleukin⁃3（IL⁃3）were screened and
optimized，in order to lay the foundation for effective culture of mouse bone marrow⁃derived mast cells（BMMCs）in vitro. Methods：
Nucleotide sequences of mouse IL⁃3 were optimized according to the commonly used codons in Escherichia coli（E. coli）and it were
then synthesized and cloned into the pET⁃28a（+）vector. The mouse IL⁃3 plasmid was transformed into E. coli BL21（DE3）and the
expression conditions was optimized. The recombinant mouse IL⁃3 was further purified and applied in the culture of mouse bone marrow
⁃derived mast cells（BMMCs）in vitro to confirm its bioactivity. Results：The plasmid of pET⁃28a（+）⁃IL⁃3 was successfully constructed，

and the recombinant mouse IL⁃3 was expressed at approximately 16 kDa by SDS⁃PAGE analysis. The finally purified mouse IL⁃3
combined with mouse stem cell factor could induce the differentiation of surface FcεRI and CD117 double positive BMMCs，which
further exhibited the function of β⁃Hexosaminidase release. Conclusion：In this study，the recombinant plasmid for mouse IL⁃3 was
constructed and the expression conditions in E.coli were optimized accordingly. The finally purified mouse IL⁃3 could be successfully
utilized in the culture of BMMCs in vitro for the investigation of allergy.
［Key words］ interleukin⁃3；mast cell；Escherichia coli；prokaryotic expression
［J Nanjing Med Univ，2023，43（02）：179⁃186］

由免疫球蛋白 E（immunoglobulin⁃E，IgE）介导 变化和环境污染加重，过敏性疾病的发病率仍在逐
的过敏性疾病是临床上的常见病、多发病，其影响 年上升 ［1-2］ 。肥大细胞是引起过敏性疾病的主要效
超过全球30%的人口，并且伴随着人们生活方式的 应细胞之一，其表面 FcεRⅠ受体结合的特异性 IgE