Preparation And Identification Of Soluble Chinese Rhesus Macaques Mamu-B*1703 Monomer And Tetramer Loaded With A SIV Peptide

AIDS and human immunodeficiency virus (HIV) infection shows an increasing trend year by year, which affects human life seriously and becomes a research hotspot in the world. HIV vaccine is the best strategy for defending virus infection and spreading. Since rhesus macaques infected with simian immunodeficiency virus (SIV) have similar pathological changes and clinical manifestation to HIV-infected human, these monkeys have become an optimal model to date for studying the pathogenesis of mankind infected with HIV and for evaluating the potential HIV vaccines. Rhesus macaques are classified as Chinese-and Indian-origin populations. There are some differences in morphology or physiology between these two populations. Since Indian-origin rhesus macaques have been prohibited from exporting, Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation.It is well known that cytotoxic T lymphocytes (CTLs) play a key role for controlling and eliminating SIV viruses. Peptides processed from the pathogens are carried by those major histocompatibility complex (MHC) molecules to the cell surface and presented to CD8+ CTLs, by which the infected cells are eventually destructed. Thus, for studying the pathogenesis of mankind infected with HIV virus and evaluating potential HIV vaccines, it is very important to analyze the SIV-specific CTL immune responses. MHC classⅠ(MHC I) tetramer technology is the "golden standard" for detecting antigen-specific CTLs, because it has high sensitivity and specificity. Since each tetramer is only used for specific MHC I and peptide, the relatively high frequency allele should be chosen for preparing a tetramer.MHC genes in rhesus macaques are named Mamu, which located on chromosome 6q24 while human MHC located on chromosome 6p21.3. Compare with human MHC I genes, rhesus macaques MHC I genes are more complex. Furthermore, there are significant differences between Chinese-and Indian-origin rhesus macaques MHC I alleles, and little is known about genetic background of Chinese-origin rhesus macaques. However, new classes of Chinese rhesus macaques MHC I allele have continually been identified in recent years. Mamu-B is an MHC I allele locus in macaques and Mamu-B*1703 become an interest molecule to us as it is a relatively high frequency allele in Chinese macaques. In this study, Mamu-B*1703 was selected for preparing a tetramer loaded with Mamu-B*1701-restricted SIV/Nef epitope (IRYPKTFGW, IW9), i.e. Mamu-B*1703/IW9 tetramer. DNA fragment encoding the ectodomain of Mamu-B*1703 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by three rounds PCR with the Mamu-B*1703 sequence cloned in pMD19-T vector as an initial template. The prokaryotic expression vector was constructed and verified by double restriction enzyme digestion and DNA sequencing. The expression vector pET-3d/Mamu-B*1703-BSP was transformed into E. coli BL21(DE3) and the recombinant protein expression was induced with IPTG. It was then refolded in the presence ofβ2-microglobulin and SIV peptide IW9 to form a soluble Mamu-B*1703 monomer. After biotinylation and purification, the tetramer was formed by incubation with streptavidin-PE at a ratio of 4:1. Its conformation was confirmed by monoclonal antibody (W6/32) recognition assay based on ELISA using HLA-A*0201 as a positive control.In conclusion, we constructed successfully a soluble Mamu-B*1703 monomer and tetramer loaded with an SIV peptide, which lay the foundation for further characterization of SIV-specific CD8+ CTLs from Chinese rhesus macaques.