title: [Expression and purification ficolin 3 and its role for RAW264.7 macrophage activation in vitro].
abstract: 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 XhoI and SalI 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-ficolin 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Nephrology, Yinchuan 750000, China.
authors.forename: Jiangtao
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Neurology, Ningxia People's Hospital, Yinchuan 750000, China.
authors.forename: Xuqing
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Radiology, General Hospital of Ningxia Medical University, Yinchuan 750000, China.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000502:physiology; D005434:Flow Cytometry; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D037102:Lectins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C109662:FCN3 protein, human; D006023:Glycoproteins; D015850:Interleukin-6; D037102:Lectins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12