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  

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

作　　者：胡昊扬 许志强 HU Haoyang;XU Zhiqiang(School of Medicine,Nantong University,Nantong 226019;Department of Pharmacy,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南通大学医学院,江苏南通226019 [2]南京医科大学第一附属医院药学部,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2023年第2期179-186,共8页Journal of Nanjing Medical University(Natural Sciences)

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p ET-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。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 p ET-28a(+)-IL-3 was successfully constructed,and the recombinant mouse IL-3 was expressed at approximately 16 k Da by SDS-PAGE analysis. The finally purified mouse IL-3combined 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.

关 键 词：白细胞介素3 肥大细胞 大肠杆菌 原核表达 

分 类 号：R329.26[医药卫生—人体解剖和组织胚胎学]