Improved expression of HLA-A* 2402-BSP in Escherichia coli and its tetramer preparation.
- Author:
Qian-Tao JIA
1
;
Li-Hui XU
;
Feng-Yao LI
;
Qing-Bing ZHA
;
Xian-Hui HE
Author Information
1. Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
CD8-Positive T-Lymphocytes;
cytology;
metabolism;
Carbon-Nitrogen Ligases;
metabolism;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
genetics;
Escherichia coli Proteins;
metabolism;
Flow Cytometry;
Gene Expression;
HLA-A Antigens;
chemistry;
genetics;
metabolism;
HLA-A24 Antigen;
Humans;
Oligopeptides;
genetics;
metabolism;
Phosphoproteins;
chemistry;
genetics;
metabolism;
Protein Multimerization;
Recombinant Fusion Proteins;
chemistry;
genetics;
metabolism;
Repressor Proteins;
metabolism;
Substrate Specificity;
T-Lymphocytes, Cytotoxic;
cytology;
metabolism;
Viral Matrix Proteins;
chemistry;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2007;23(2):284-291
- CountryChina
- Language:Chinese
-
Abstract:
HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.
