[Improved expression of HLA-A* 2402-BSP in Escherichia coli and its tetramer preparation].
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.
Jia
Qian-Tao
QT
Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Xu
Li-Hui
LH
Li
Feng-Yao
FY
Zha
Qing-Bing
QB
He
Xian-Hui
XH
D000595:Amino Acid Sequence; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D015234:HLA-A Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D059871:HLA-A24 Antigen; D006801:Humans; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D015234:HLA-A Antigens; C438506:HLA-A*24:02 antigen; D059871:HLA-A24 Antigen; D009842:Oligopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli