Primary Refolding Of The HLA-A～*0201-peptide Complexes And Its Application In HBV Variation

The heavy chain of human histocompatibility leukocyte antigen (HLA)-A*0201 and the light chain (p2-microglobulin,p2m) were amplified from cell lines CIR/A0201 and Raji by RT-PCR respectively. A 15-amino acid (aa) substrate peptide for BirA-dependent biotinylation (BSP) was added to the COOH-terminus of heavy chain at aa275 by PCR to make the HLA-A*0201 -peptide complexes have higher affinity and be more suitable for use as an immunologic stain labelled with streptavidin. After the sequences were confirmed, the DNAs of P2m and heavy chain-BSP were digested with Ndel/sap I and Xbal /Sap I respectively, and ligated into pTYB 1 vector which had been digested with the corresponding enzymes. The plasmids were transformed into Escherichia coli 2566, a heavy chain-BSP producing clone and a P2m producing clone upon induction with isopropyl p-D-thiogalactopyranoside(IPTG) were submitted to DNA sequencing to verify their sequence. Heavy chain-BSP-intein fusion protein and P2m- intein fusion protein have been expressed at high levels in the form of inclusion body in bacterial cells, and peptides of P2m and heavy chain-BSP were purified by removal of the intein in the presence of DTT on a chitin column from BioLabs. Only in the presence of an HLA-A*0201-restricted peptide such as HBcAgl8-27 was a stable HLA-A*0201 complexes formed in vitro, which lays the foundation for further constructing HLA-A*0201 tetramers.No influence of hepatitis B virus (HBV)/C gene spot mutation S87G and/or I97L on HLA-A*0201 -restricted cytotoxic T lymphocyte epitopes by application of T cell epitope predicting computer program in combination with the formational character of HLA-A*0201 tetramers in vitro.