The Preliminary Research On Reconstruct Hla-ⅰto Identify SARS-COV T Cell Epitopes In Vitro

Major histocompatibility complex (MHC) is a group of closely linked genes in chromosome of mammal. It is the key element of antigen presentation and T cell activation. Severe acute respiratory syndrome (SARS) is a high pathogenicity, high prevalence and high mortalityrate rate of new human infection diseases. S protein plays an important role in virus and cell membrance fusion and anti-viral immunity of organism. Studies on SARS-COV mostly depend on the development of animal models, while the MHC-peptide polymer technology can identify the epitope of SARS and can conduct preliminary and reliable evaluation of vaccine. The prediction of S protein T cell epitope showed that the S protein had high affinity binding with antigen epitope with MHC(HLA) and suggested this region plays an important role in stimulating the body's immune response. In this study, we used reconstruction of HLAⅠmolecule comllex to indetified four of SARS-COVS protein T cell epitope.1. Two pair of primers were designed according to reference extracellular domain of HLA-Aαchain mature peptide gene sequeces and Humanβ2m chain mature peptide gene sequeces published in GenBank. The extracellular domain gene was amplified from the blood of healthy people by using RT-PCR. PCR product was cloned respectively into the T easy vector and sequenced. The sequencing result showed that HLA-Aαchain mature peptide gene sequeces was 819 bp and encoding a peptide of 273 amino acids whose molecular is about 31.45KDa, hydrophobic amino acid is 26.53%, hydrophilic amino acid is 28.83%, basic amino acid is 15.27% and acidic amino acid is 15.12%. The homology between extracellular domain of HLA-Aαchain mature peptide gene sequeces and the template is 96%. Compared with the mature peptide gene sequence of HLA-Aαchain with those of cattle, chicken, dog, duck, frog, goose, Grass carp, human, horse, mouse, Orangutan, Quail, Rhesus monkey and Swine, showed that the nucleotides homology were 76.9%,38.5%,81.9%,39.1%,31.4%,39.3%,29.7%,93%,81%,68.1%,83.5%,39.5%,81% and 76.6% respectively. The Humanβ2-microglobulin chain mature peptide gene sequeces was 297 bp and encoding a peptide of 99 amino acids whose molecular is about 11.73KDa, hydrophobic amino acid is 28.15%, hydrophilic amino acid is 29.24%, basic amino acid is 15.40% and acidic amino acid is 15.67%. The gene sequence was 100% homology withβ2-microglobulin chain mature peptide on Genebank. Compared with nucleotides sequence of mature peptide gene sequences ofβ2m chain with those of cattle, chicken, fish, human, Rhesus monkey, mouse, Ornithorhynchus, Pongo and sheep, showed that the nucleotides homology were 73.5%,45.5%,47.4%,100%,90.9%,69.7%,53.6%,100% and 73.5%.2. In order to express successfully extracellular mature peptide gene of HLA-Aαchain andβ2m chain in E.coli. Two pair of primers was designed respectively and the extracellular mature peptide gene of HLA-Aαchain andβ2m chain was subcloned into the prokaryotic expression vector of pET-28 a (+) and transformed into BL21. After induced by IPTG, the expression of extracellular mature peptide gene of HLA-Aαchain andβ2m chain was detected by SDS-PAGE. The result showed that the extracellular mature peptide gene of HLA-Aαchain andβ2m chain gene could be highly efficientm and stable expression in E. coli.3. These four peptides were selected respectively with HLA-Aαand HLA-β2m folded together refolding and renaturation in vitro. Refolding and renaturation could back to its original conformation space and made epitope, HLA-Aαand HLA-β2m folding into the HLA-A/ epitope monomer complex. Non-denaturing SDS-PAGE showed that HLA-Aα, HLA-β2m and SP4 folded successfully into HLA-A/SP4 monomer complex. MHCⅠmolecules was successfully expressed in prokaryotic expression system and folded in vitro that provides a more intuitive method of MHC I molecules binding peptide in vitro, this method provides a theoretical reference for the genetic engineering vaccine preparation.