Identification Of Monoclonal Antibodies Against The Epitopes On SHIV P27 Capsid Protein

Chimeric simian/human immunodeficiency virus (SHIV) was developed by replacing the simian immunodeficiency virus (SIV) envelope protein gene (env) and auxiliary genes with corresponding genes from human immunodeficiency virus (HIV-1). SHIV has therefore obtained some biological and immunological characteristics from HIV-1, and SHIV-infected macaques have been applied as non-human primate (NHP) models for studies on basic researches and vaccine development of AIDS.The SHIV p27 capsid protein forms the inside core of the double-shell virions, and is one of the major sources of immunogens of SHIV. Therefore, p27 and p27 antibodies are frequently used to monitor the infection of SHIV in vitro and in vivo for studies on vaccine development, concept of immunization strategies and basic researches of AIDS and HIV-1. It is important to develop monoclonal antibodies against p27 and to identify binding epitopes of p27 antibodies for further studies on the capsid and for the development of detection and diagnostic reagents of SHIV.Three p27 monoclonal antibodies (MAbs), p27-A5, p27-C7 and p27-D2, were developed in a previous study of this laboratory. In this study, these mAbs were firstly concentrated and purified by using a Protein A/Sepharose CL-4B chromatography. The binding sites of these three p27 mAbs were then evaluated by synergic ELISA using different combination of these mAbs as the primary detecting antibody. Results suggested that these three mAbs recognized two different binding sites. To locate these binding sites more accurately, the p27 cDNA was subcloned as three individual fragments with a 15-bp overlapping between each others. These fragments were named as p27-a (1bp-255bp), p27-b (241bp-495bp) and p27-c (481bp-741bp). As for the subcloning, p27 fragments were amplified by PRC using primers with BamH I and Hind III restriction sites at each ends respectively for fragments of p27-a,p27-b and p27-c. All these amplified fragments were cloned into a prokaryotic expression vector pET-32a after being registered by corresponding restriction endonucleases and were checked for correct insertion by restriction enzyme analysis and sequencing.P27 polypeptides were expressed by these vectors and the products were confirmed being able to react with p27 mAbs by Western blot. Based on these results, a set of sixteen 16-residue peptides were designed to cover the whole immunopredominant region of p27, which were overlapped at both ends each other and named as A1-A8 and C1-C8. To express these peptides as fusion proteins with a His-tag, corresponding DNA fragments were synthesized with restriction sites BamH and Xho I at the 5'and 3'ends, respectively, and were subcloned into the expression vector pET32. The resultant clones were accordingly termed as pET-32a-A1 to ET-32a-A8 and pET-32a-C1to pET32a-C8. This set of p27 fusion peptides expressed by these plasmids in E. coli was further checked by SDS-PAGE and Western blot and were used to react with the three p27 mAbs for locating binding sites of these antibodies.The following results were obtained in this study:1. Three anti-p27 monoclonal antibodies were purified and concentrated, which are ready for future experiments and studies.2. It was identified that mAbs p27-A5 and p27-C7 recognized the identical or similar epitope, and p27-D2 bound a different one at a relatively separated location in p27. 3. The detailed binding sites of these three p27 mAbs were further identified, of which mAbs p27-C7 and p27-A5 reacted to the p27 residues 61-76 with the sequence of 61SEGCTPYDINQMLNCV76, and the mAb p27-D2 targeted to residues located at 191-206 with the sequence of 191EQTDAAVKNWMTQTLL206.In conclusion, the reaction epitops of three p27 mAbs in the target protein were identified in this study. Furthermore, several experimental techniques and protocols were established, which include cloning and expression of target gene fragments in E. coli protein detection, antibody purification and detection of antigen/antibody interactions. The immunologically characterized p27 mAbs will be used to develop detection kits for SHIV infection and replication, which are important assays for AIDS related studies.