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Updated: Aug 8, 2026
Generation of Human CD40-activated B cells
Published on: October 17, 2009
Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory
1Institute of Immunopathology, Xi'an Medical University, 205 Scarlet Bird Street, Xi'an 710061, China.
Insights
Bacterially produced human B7-1, a functional glycoprotein, requires both its immunoglobulin-like domains for receptor binding. This study identified that the complete extracellular region is essential for T cell activation.
Area of Science:
- Immunology
- Molecular Biology
- Protein Expression
Background:
- The human B7-1 molecule plays a crucial role in T cell co-stimulation.
- Understanding the specific domains responsible for B7-1 receptor binding is essential for elucidating its function.
Purpose of the Study:
- To determine which immunoglobulin (Ig)-like domain, IgV or IgC, of human B7-1 contains the receptor binding sites.
- To assess the biological activity of human B7-1 expressed in bacterial systems.
Main Methods:
- Polymerase Chain Reaction (PCR) was used to amplify IgV, IgC, and combined IgV+IgC domains of human B7-1.
- Recombinant plasmids were constructed and introduced into E. coli (M15 strain) for protein expression.
- SDS-PAGE, Western blotting, and T lymphocyte activation assays (using anti-CD3 antibody and [3H]-TdR incorporation) were employed to analyze protein products and biological activity.
Main Results:
- Three recombinant human B7-1 proteins (hB7-1 IgV, hB7-1 IgC, and hB7-1 IgV+IgC) were successfully produced in bacterial cells in both soluble and inclusion body forms.
- Co-stimulation with bacterially produced hB7-1 (IgV+IgC) induced T lymphocyte proliferation in the presence of anti-CD3 antibody.
- Neither the isolated hB7-1 IgV nor hB7-1 IgC domains alone stimulated T lymphocyte proliferation.
Conclusions:
- Functional human B7-1 glycoprotein can be produced in bacterial expression systems.
- Both extracellular immunoglobulin-like domains (IgV and IgC) of human B7-1 are necessary for its interaction with counter receptors and subsequent T cell activation.
Objective:
To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity.
Methods:
PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC). Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15. The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and [3H]-TdR incorporation.
Results:
Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells. With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC.
Conclusions:
Functional glycoprotein human B7-1 could be produced in bacterial cells. Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.
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