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

doi:10.13523/j.cb.20180201

China Biotechnology

2018, Vol. 38

Issue (2): 1-6 DOI: 10.13523/j.cb.20180201

Orginal Article

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(

)

School of Basic Medical Immunology, Hubei Province Key Laboratory of Allergy and Immunology,and Department of Immunology, State Key Laboratory of Virology, Medical Research Institute,Wuhan University, Wuhan 430071, China

Download:

HTML

PDF(717KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective： CD19 scFv (single-chain fragment variable) specifically recognizes CD19 molecule expressed on the B cells surface. Interleukin-10 (IL-10) -producing B cells are a major subset of regulatory B cells (Breg). The aim is to construct a recombinant fusion protein of CD19 scFv and IL-10 receptor1 (IL-10R1), which potentially captures and blocks IL-10 secreted by Breg cells.Methods: CD19scFv and IL-10R1 were cloned into pET-28a prokaryotic plasmid. Recombinant proteins were enriched and purified by Ni column, identified by SDS-PAGE and Western blot. Pull down analysis was performed to identify the proteins captured by CD19scFv-IL-10R1 recombinant protein.Results: The CD19 scFv-IL-10R1 fusion protein is prepared and the protein binds to both CD19 and IL-10 molecules in vitro.Conclusion: CD19scFv-IL-10R1 fusion protein binds to both CD19 molecule and IL-10 molecule in vitro, and has potential application as a specific inhibitor for Bregs.

Key words： IL-10 Regulatory B cells IL-10R1 CD19-scFv

Received: 28 July 2017 Published: 21 March 2018

ZTFLH:

Q819

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Rong ZHAO
	Han-yu CHEN
	Chun HUANG
	Xiao-lian ZHANG
	Qin PAN

Cite this article:

Rong ZHAO,Han-yu CHEN,Chun HUANG,Xiao-lian ZHANG,Qin PAN. Construction, Expression and Identification of Recombinant Fusion Protein Specifically Targeting B Cells and Binding to IL-10. China Biotechnology, 2018, 38(2): 1-6.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180201 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I2/1

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

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


[1]	Sabat R, Grutz G, Warszawska K , et al. Biology of interleukin-10. Cytokine Growth Factor Rev, 2010,21(5):331-344. doi: 10.1016/j.cytogfr.2010.09.002

[2]	Yanaba K, Bouaziz J D, Haas K M , et al. A regulatory B cell subset with a unique CD1d hi CD5 + phenotype controls T cell dependent inflammatory responses . Immunity, 2008,28(5):639-650. doi: 10.1016/j.immuni.2008.03.017 pmid: 18482568

[3]	Jurlander J, Lai C F, Tan J , et al. Characterization of interleukin-10 receptor expression on B-cell chronic lymphocytic leukemia cells. Blood, 1997,89(11):4146-4152. doi: 10.1007/s002770050304 pmid: 9166857

[4]	Carter R H, Fearon D T . CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science, 1992,256(5053):105-107. doi: 10.1126/science.1373518 pmid: 1373518

[5]	Yuan-Qin Min . Targeting of envelope protein E2 antigen to B cells via CD19-Scfv for HCV vaccine design. Wuhan:Wuhan University, 2013,1.

[6]	Ma Y, Xiang D, Sun J , et al. Targeting of antigens to B lymphocytes via CD19 as a means for tumor vaccine development. Immunol, 2013,190(11):5588-5599. doi: 10.4049/jimmunol.1203216 pmid: 23630363

[7]	Yanaba K, Bouaziz J D, Matsushita T , et al. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals. Immunol, 2009,182(12):7459-7472. doi: 10.4049/jimmunol.0900270

[8]	Schlom J . Therapeutic cancer vaccines: current status and moving for-ward. Natl Cancer Inst, 2012,104(11):599-613. doi: 10.1093/jnci/djs033 pmid: 3328421

[9]	Zhang Y, Eliav Y, Shin S U , et al. B lymphocyte inhibition of anti-tumor response depends on expansion of Treg but is independent of B-cell IL-10 secretion. Cancer Immunol Immun, 2013,62(1):87-99. doi: 10.1007/s00262-012-1313-6 pmid: 22772949

[10]	Olkhanud P B, Damdinsuren B, Bodogai M , et al. Tumor evoked regulatory B cells promote breast cancer metastasis by converting resting C D 4 + T cells to T-regulatory cells . Cancer Res, 2011,71(10):3505-3515. doi: 10.1158/0008-5472.CAN-10-4316 pmid: 3096701

No related articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed