title: [The expression of human IL-1β gene containing human erythropoietin signal peptide in HepG2 cells].
abstract: OBJECTIVE
  To construct two lentiviruses secreting human IL-1β through either classical or nonclassical pathway and analyze their expressions in HepG2 cells after packaging lentiviruses and infecting hepatoma carcinoma HepG2 cells.
  
  
  METHODS
  Human full-length IL-1β gene and chimeric gene containing human erythropoietin(EPO) signal peptide and mature IL-1β protein coding sequence were respectively amplified from pIRES2-EGFP-proIL-1β and pIRES2-EGFP-epoIL-1β using PCR. The sequences were subsequently cloned into lentiviral expression vector pLenti6/V5 to construct pLenti6/V5-proIL-1β, which expressed IL-1β through nonclassical pathway, and pLenti6/V5-epoIL-1β, which expressed IL-1β through signal-peptide mediated classical pathway. Lentiviruses expressing human IL-1β through either classical or nonclassical pathway were packaged in HEK293T cells using a three-plasmid packaging system, and then these viruses were used to infect HepG2 cells. The level of IL-1β in both cytoplasm and culture supernatant were detected by sandwich ELISA and Western blotting.
  
  
  RESULTS
  pLenti6/V5-proIL-1β expressing human full-length IL-1β gene and pLenti6/V5-epoIL-1β expressing human EPO signal peptide and mature IL-1β gene were successfully constructed and confirmed through enzymatic assay and DNA sequencing. The lentiviruses expressing IL-1β through different pathways were prepared using a three-plasmid packaging system in HEK293T cells. Compared with the cells infected with control virus, levels of supernatant and cytoplasmic IL-1β in the cells infected with two lentiviruses expressing IL-1β through different pathways were markedly elevated (P<0.01). However, level of mature IL-1β in supernatant of HepG2/epoIL-1β cells was much higher than that of HepG2/proIL-1β cells, while total IL-1β level in cytoplasm of HepG2/proIL-1β cells was significantly higher than that in HepG2/epoIL-1β cells.
  
  
  CONCLUSIONS
  Both classical and nonclassical pathway secretion vectors could express human IL-1β in HepG2 cells, but EPO signal-peptide mediated classical pathway secreted much higher mature IL-1β than that of nonclassical pathway.
authors.affiliation: Key Laboratory for Immunology in Universities of Shandong Province, Department of Immunology, Weifang Medical University, Weifang 261053, China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Key Laboratory for Immunology in Universities of Shandong Province, Department of Immunology, Weifang Medical University, Weifang 261053, China.
authors.forename: Dalin
authors.initials: D
authors.lastname: DI
authors.affiliation: Key Laboratory for Immunology in Universities of Shandong Province, Department of Immunology, Weifang Medical University, Weifang 261053, China.
authors.forename: Weiling
authors.initials: W
authors.lastname: Xiao
authors.affiliation: Key Laboratory for Immunology in Universities of Shandong Province, Department of Immunology, Weifang Medical University, Weifang 261053, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Fu
authors.affiliation: Key Laboratory for Immunology in Universities of Shandong Province, Department of Immunology, Weifang Medical University, Weifang 261053, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Liang
mesh_terms: D015153:Blotting, Western; D017077:Culture Media, Conditioned / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D019065:Virus Assembly
chemical_list: D017077:Culture Media, Conditioned; D053583:Interleukin-1beta; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin