China Biotechnology

Construction, Expression and Identification of Recombinant Fusion Protein Specifically Targeting B Cells and Binding to IL-10

Rong ZHAO,Han-yu CHEN,Chun HUANG,Xiao-lian ZHANG,Qin PAN

China Biotechnology, 2018, 38(2): 1-6. DOI: 10.13523/j.cb.20180201

Fig.1 Construction of pET-28a-CD19scFv-IL-10R1 plasmid

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Fig.2 Identification of PCR products of CD19scFv and IL-10R1 gene fragments(a) Agarose gel electrophoresis analysis of PCR product of CD19scFv (b) Agarose gel electrophoresis analysis of PCR product of IL-10R1
Fig.3 Identification of pET-28a-CD19scFv plasmid (a)and pET-28a-CD19scv-IL-10R1 plasmid (b)(a) pET-28a-CD19-scfv plasmid was treated with BamHI and HindIII 1: pET-28a; 2: Digestion with BamHI and HindIII (b) 1: pET-28a; 2: Digestion with BamHⅠ and XhoⅠ
Fig.4 Expression of CD19scFv protein at 25℃
Fig.5 Expression of CD19scFv-IL-10R1 protein at different temperatures and different IPTG concentrations(a) CD19scfv-IL-10R1 was expressed at 30 ℃ and (0.4mmol/L~2mmol/L) IPTG concentrations (b) CD19scfv-IL-10R1 was expressed at 25 ℃ and (0.4mmol/L~2mmol/L) IPTG concentrations (c) CD19scfv-IL-10R1 was expressed at 16 ℃ and (0.4mmol/L~2mmol/L) IPTG concentrations (d) CD19scfv-IL-10R1 was expressed at 4 ℃ and (0.4mmol/L~2mmol/L) IPTG concentrations
Fig.6 Determination of purified CD19scFv and CD19scFv-IL-10R1 recombinant protein by SDS PAGE and Western blot(a) SDS PAGE (b) Western blot
Fig.7 CD19-scfv-IL-10R1 binds to both CD19 and IL-10CD19scfv-IL-10R1/ CD19scFv-beads were incubated with cell lysate from LPS-treated B splenocytes. After washing the beads, Western blot was performed to identify the proteins captured by CD19scfv-IL-10R1/ CD19scFv-beads