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Construction and Expression of HLA-A~0206-BSP and-A~0207-BSP

Author: ZhuLiJun
Tutor: WuXiongWen
School: Huazhong University of Science and Technology
Course: Immunology
Keywords: Clone HLA-A ~ * 0206 and -A * 0207 Biotinylated sequence
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 18
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Abstract

Background: The major histocompatibility molecules (MHC) plays a very important role in the immune response, its biological function is presented to T cell recognition of antigen peptides. MHC molecules are highly polymorphic, the polymorphism of amino acid residues is often located in antigenic peptide binding groove, so that the type of MHC molecules do not affect the binding and presenting antigen peptide, thus affecting the T cell response. HLA-A2 is the most common HLA class I molecules I type HLA-A2 positive individuals accounted for about 50% of our population. antigen peptide spectrum of subtypes molecular recognition, affinity, and the induction of cytotoxic T lymphocytes (cyto toxic T lymphocyte, CTL) response capability with a wide range of practical significance in recent years, cases of observed data from foreign clinical transplantation prompted for by by HLA-A2 subtypes match can significantly improve survival and effect of transplantation of hematopoietic stem cell transplant recipients [3]. Objective and Methods: In order to clone the HLA-A * 0206-and-A * 0207 cDNA sequence, to construct and express the HLA-A * 0206 and-A * 0207 α chain (heavy chain) extracellular region and the BirA enzyme substrate peptide (BirA substrate peptide, BSP) of the fusion protein. In this study, using RT-PCR technology from HLA-A * 0206-A * 0207-positive individuals in PBMC were cloned HLA-A * 0206-A * 0207 full-length cDNA sequence, build the HLA-A * 0206 and-A * 0207 cloning vector. HLA-A * 0206 and-A * 0207 α chain (heavy chain) cellular outer segment sequence using PCR amplification from the constructed cloning vector, respectively, was replaced by the double digestion stored in our laboratory, HLA-A * 0201 - BSP recombinant HLA-A * 0201 extracellular domain sequences, so that an HLA-A * 0206-and-A * 0207 with the BirA enzyme substrate peptide (birA SUBSTRATE peptide, the BSP) sequence fused to construct HLA-A * 0206 and - A * 0207-BSP fusion gene expression vectors. Then the expression vector was transformed into E coli BL21 (DE3) expression product by in vitro refolding, preliminary purified expression products detected by ELISA and WESTERN-BLOT. In order for further construction of the HLA-A * 0206-A * 0207 four-mer or coated microspheres made of artificial antigen-presenting cells, features provides the material basis of the corresponding HLA-A2 subtypes. Results: successful amplification of full-length cDNA sequences of the HLA-A * 0206-A * 0207 heavy chain and its extracellular domain sequence, build the HLA-A * 0206-BSP-A * 0207-BSP fusion gene expression vector and confirmed by restriction enzyme digestion, PCR and DNA sequencing; expression product of the expression vector was transformed into E. coli BL21 (DE3), through in vitro refolding preliminary purified expression product with β2 microglobulin (HLA class I molecule light chain) and the HLA-A2-restricted antigen peptides (HBV core 18-27) folded to form the antigenic peptide / HLA-A2 complex monomer having the natural conformation of the HLA class I molecule. Conclusion: This study on the basis of cloning of HLA-A * 0206-and-A * 0207 heavy chain length cDNA construct an HLA-A * 0206--BSP and-A * 0207-BSP fusion gene expression vector for further construction HLA-A * 0206-A * 0207 tetramer features provide the material basis of the corresponding HLA-A2 subtypes.

Construction and Expression of HLA-A~*0206-BSP and-A~*0207-BSP

Abstract

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Construction and Expression of HLA-A~0206-BSP and-A~0207-BSP