Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro.
OBJECTIVE
To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.


METHODS
Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), 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 B7.2 fusion proteins by [(3)H]-TdR incorporation.


RESULTS
Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.


CONCLUSIONS
Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.
Yan
Xiaocai
X
Insitute of Immunopathology, Medical School of Xi'an Jiaotong Univerisity, Xi'an 710061, China.
Ma
Jun
J
Zheng
Jin
J
Lai
Baochang
B
Geng
Yiping
Y
Wang
Yili
Y
Si
Lüsheng
L
D015703:Antigens, CD / Q000494:pharmacology*; D051940:B7-2 Antigen; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000494:pharmacology*; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000276:immunology
D015703:Antigens, CD; D051940:B7-2 Antigen; C497964:CD86 protein, human; D007127:Immunoglobulin Constant Regions; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins