6 Histidine Specific Recognition by Variable Lymphocyte Receptors(VLRs)

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2012. 2. 한병우.Lamprey and hagfish are the only two surviving jawless vertebrates. Instead of immunoglobulin superfamily folds, these jawless vertebrates adopt variable lymphocyte receptors (VLRs) for antigen recognition in the adaptive immune system. VLRs are assembled by genetic rearrangement with flanking leucine-rich repeat (LRR) cassettes and incomplete VLR genes to achieve the required repertoire for any antigens encountered. Mature VLR genes encode an N-terminal LRR capping region (LRRNT), the first LRR (LRR1), up to seven 24-residue variable LRRs (LRRVs), a terminal or end LRRV (LRRVe), a connecting peptide (CP), a C-terminal LRR capping region (LRRCT), and threonine/proline-rich stalk region that connects the protein to a glycosylphosphatidylinositol (GPI) anchor and hydrophobic tail. Since antibodies are known to recognize most of foreign antigens and monoclonal antibody has been used for diagnostic applications, research tools, and therapy, VLRs would also be a promising monoclonal antibody alternative. Especially, VLRs which specifically recognize 6 histidine peptide could be used for research tools such as immunoaffinity purification of novel fusion proteins, protein chip, flow cytometry and immunohistochemistry. To elicit 6 histidine specific VLRs, the hagfish was immunized with 6 histidine peptide for two months with two week interval. mRNA was purified from their blood and cDNA was generated by RT-PCR. VLR libraries that are expected to recognize 6 histidine peptides specifically were constructed and VLR proteins were overexpressed with maltose binding protein (MBP) fusion using E.coli system. I purified the proteins by affinity chromatography, ion exchange chromatography, and size-exclusion chromatography. To detect VLRs specifically, anti-VLR monoclonal antibody was elicited with mice and the anti-VLR monoclonal antibody recognizes well-conserved threonine/proline-rich stalk region of VLRs. To screen 6 histidine specific VLRs efficiently, ELISA experiments with the elicited anti-VLR monoclonal antibody are in progress. This research has its own unique advantages over conventional monoclonal antibody technologies or previous VLR studies.Maste