摘要
目的构建人ficolin-3基因的原核表达载体在大肠杆菌中表达后纯化,检测可溶性His-ficolin在诱导巨噬细胞系RAW264.7活化中的作用。方法从人外周血单个核细胞cDNA中扩增人ficolin-3基因的cDNA序列,将其插入pET22原核表达载体中,酶切鉴定并测序验证重组质粒pET22b-ficolin 3后,在大肠杆菌BL21中诱导表达并纯化His-ficolin 3。以His蛋白为对照,利用流式细胞术检测不同浓度可溶性His-ficolin 3对RAW264.7细胞吞噬鸡红细胞能力的影响,利用ELISA检测His-ficolin 3对RAW264.7细胞分泌IL-6、IL-12和TNF-α的影响。结果酶切鉴定获得预期目的片段,测序证实重组质粒pET22b-ficolin 3构建成功。SDS-PAGE提示相对分子质量(Mr)33 000处有可溶性目的蛋白His-ficolin 3表达,His柱纯化后产物纯度在90%以上。Western blot法检测证实纯化产物为His-ficolin 3。和His蛋白相比,His-ficolin 3对RAW264.7细胞的吞噬能力具有剂量依赖性,即His-ficolin3浓度越高,吞噬能力越强;此外,His-ficolin3能显著诱导RAW264.7细胞分泌IL-6、IL-12和TNF-α等炎性细胞因子。结论成功构建了pETb-Ficolin 3重组载体并获得了纯度为90%以上的His-ficolin 3。His-ficolin 3能够显著促进巨噬细胞RAW264.7的活化。
Objective To establish a prokaryotic expression plasmid of ficolin-3, express and purify the fusion protein His-ficolin 3 in E. coli, and explore the role of ficolin-3 in the activation of RAW264.7 macrophages. Methods Using the cDNA from human peripheral blood mononuclear cells as template, human ficolin-3 cDNA was amplified by PCR and inserted into vector pET22b. The recombinant plasmid pET22b-ficolin 3 was identified by digestion of Xho I and Sal I and confirmed by DNA sequencing. Thereafter, the protein His-ficolin 3 was induced and purified in E. coli BL21. His-fincolin 3 at different concentrations was added into RAW264.7 cells. With the protein His as control, the effects of His-fincolin 3 on the phagocytosis of RAW264. 7 cells to chicken red blood cells （CRBCs） and on the production of IL-6, IL-12 and TNF-α were tested by flow cytometry and ELISA, respectively. Results Enzyme digestion and sequencing confirmed that recombinant plasmid pET22b-ficolin 3 was established as expected. SDS-PAGE showed the expression of soluble His-flcolin 3 of Mr 33 000. The purity of the products was over 90%. Western blotting further identified His-ficolin 3. Compared with His protein, His-ficolin 3 improved the phagocytosis of RAW264.7 cells in a dose-dependent manner, and it raised the secretion of IL-6, IL-12 and TNF-α in RAW264.7 cells. Conclusion The plasmid pETb-ficolin 3 was cloned successfully and the purity of the protein His-ficolin 3 was over 90%. His-ficolin 3 could induce the activation of RAW264.7 cells obviously.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第7期677-680,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
宁夏自然科学基金(NZ11157)
宁夏科技攻关项目(2009032)
