Soluble HLA A 0201 PR1 Construction and Identification of Complex.docVIP

 PAGE \* MERGEFORMAT 25 Soluble HLA A 0201 PR1 Construction and Identification of Complex Author: Wan-Jun Sun Xu Gang Hu Hailan Zou Min-Ji Jian-Fang Du WANG Jin-feng CAI Xin Jia Xi Hui-Sheng Ai Abstract prepared in HLA  A * 0201  PR1 tetramer, the subjects constructed soluble HLA  A * 0201  PR1 complex. The original expression of HLA  A * 0201  BSP fusion protein for the heavy chain, β2  microglobulin (β2 m) for the light chain, with the synthetic HLA  A2 restricted peptide PR1 (proteinase 3, the first 169  177 amino acids, VLQELNVTV) for a total re-folded, so as to produce soluble HLA  A * 0201  PR1 complex. Application of BirA enzyme refolding mixture to biotin and then purified by anion-exchange column obtained biotinylated soluble HLA  A * 0201  PR1 complex. Application of gel filtration high-performance liquid chromatography analysis of soluble HLA  A * 0201  PR1 complex formation rate. The use of HLA  A2 conformation-specific antibodies (BB7.2) and streptavidin-biotin for Western blot and ELISA analysis, biotinylation of soluble HLA  A * 0201  PR1 complexes were identified. The results showed that: In the folding system, containing mainly HLA  A * 0201  BSP polymer, HLA  A * 0201  PR1 complexes and β2 m, which is generated soluble compound rate of about 18%. Received HLA  A * 0201  PR1 complexes can be effectively under the action of BirA enzymatic biotinylation. Conclusion: The success in obtaining soluble biotinylated HLA  A * 0201  PR1 complex, for further preparation of HLA  A * 0201  PR1 tetramer created the foundation. Keywords: HLA  peptide complexes; HLA  A * 0201  PR1 complexes; HLA  A * 0201  PR1 tetramer Construction and Characterization of Soluble HLA  A * 0201  PR1 Complex Abstract This study was aimed to construct the soluble HLA  A * 0201  PR1 complex for preparation of HLA  A * 0201  PR1 tetramer. The recombinant HLA  A * 0201  BSP (BirA substrate peptide) fusion