[Homology modeling and eukaryotic expression of a modified αβ TCR harboring the immunoglobulin-like domain of γδ TCR].

Publisher: Xi bao yu fen zi mian yi xue za zhi bian ji bu Country of Publication: China NLM ID: 101139110 Publication Model: Print Cited Medium: Print ISSN: 1007-8738 (Print) Linking ISSN: 10078738 NLM ISO Abbreviation: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi Subsets: MEDLINE

Original Publication: Xiʹan : Xi bao yu fen zi mian yi xue za zhi bian ji bu

Receptors, Antigen, T-Cell, alpha-beta/*immunology ; Receptors, Antigen, T-Cell, gamma-delta/*immunology ; Recombinant Fusion Proteins/*immunology ; T-Lymphocytes/*immunology; Amino Acid Sequence ; Eukaryotic Cells/immunology ; Eukaryotic Cells/metabolism ; Gene Expression/immunology ; Humans ; Jurkat Cells ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Microscopy, Confocal ; Models, Molecular ; Protein Domains ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; Receptors, Antigen, T-Cell, alpha-beta/metabolism ; Receptors, Antigen, T-Cell, gamma-delta/genetics ; Receptors, Antigen, T-Cell, gamma-delta/metabolism ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/metabolism ; Sequence Homology, Amino Acid ; T-Lymphocytes/metabolism

Objective To design, construct and express a chimeric αβ TCR harboring the immunoglobulin-like (Ig) domain of γδ TCR in Jurkat T cells. Methods The fusion sites of TCR δIg were determined by bioinformatics analysis. Then the protein structures of TCR α δIg and TCR β δIg were predicted by homology modeling. Furthermore, the structures of TCR α δIg and TCR β δIg were compared with the wild type (wt) TCR α and TCR β respectively by combinatorial extension (CE). After that, the TCR α δIg and TCR β δIg were fused to fluorescent protein ECFP and EYFP respectively via the overlap PCR, and then the fusion genes (TCR α δIg-ECFP and TCR β δIg-EYFP) were cloned into pIRES2-EGFP vector and respectively located at the upstream and downstream of an internal ribosome entry site (IRES). The recombinant prokaryotic expression vector pIRES-TCR βδIg-EYFP/TCR αδIg-ECFP was transferred into Jurkat T cells. Finally, the expression of TCR δIg in Jurkat T cells was monitored by confocal laser scanning microscopy (CLSM). Results The variable region structure of the TCR δIg did not change and the antigen recognition active regions remained stable compared to the wtTCR. The recombinant expression plasmid was successfully constructed as confirmed by PCR identification and sequencing analysis. CLSM showed that TCR δIg was expressed and located at the plasma membrane of Jurkat T cells. Conclusion The design of TCR δIg was reasonable and the TCR δIg could be expressed on Jurkat T cell surface.

0 (Luminescent Proteins)
0 (Receptors, Antigen, T-Cell, alpha-beta)
0 (Receptors, Antigen, T-Cell, gamma-delta)
0 (Recombinant Fusion Proteins)

Date Created: 20160715 Date Completed: 20170113 Latest Revision: 20170113

20240829

27412930