Preparation of H-2Db tetramer and its application in enumerating the CD8+ T cells specific for lymphocytic choriomeningitis virus / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 278-284, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-276127
ABSTRACT
Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db KAV tetramer reagent was verified by detecting the CD8+ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8+ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining (< or = 0.01%) of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Histocompatibility Antigens Class I
/
H-2 Antigens
/
Immunologic Techniques
/
CD8-Positive T-Lymphocytes
/
T-Cell Antigen Receptor Specificity
/
Allergy and Immunology
/
Genetics
/
Lymphocytic choriomeningitis virus
/
Mice, Inbred C57BL
/
Antibody Specificity
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article
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