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| Obtaining Active Segments in Chicken (Gallus gallus) B-LB Molecule to Bind with Invariant Chain by Pull-down Method |
| 1,1, 1,1, 1,1, 1 |
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Abstract This work focus on clearing active segments of chicken (Gallus gallus) major histocompatibility complex (MHC) class Ⅱmolecules binding with invariant chain (Ii) for research of mechanism of MHC and Ii action in presenting antigen peptides. To this end 3 DNA segments (Sβ1, β1 and β2TC) were cloned from cDNA of B-LB gene, which was kept in our laboratory, and inserted into prokaryotic or eukaryotic expression plasmids respectively. Then these recombinant plasmids were respectively transfected or co-transfected with Ii into engineering bacteria, Rosetta (DE3). The expression products or complexes (B-LB segment and Ii) were purified by affinity chromatography and identified by pull-down and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the expression and localization of B-LB segments and Ii in the eukaryotic cells were observed too. The results showed that first the 3 recombinant plasmids (pGEX-4T-1-B-LB-Sβ1, pGEX-4T-1-B-LB-β1 and pGEX-4T-1-B-LB-β2TC) could well express, and the interest proteins (B-LB-Sβ1, B-LB-β1 and B-LB-β2TC) also well be purified by an affinity chromatography. Secondly the segment B-LB-Sβ1 or B-LB-β1 rather than B-LB-β2TC bound His/Ii, which adsorbed to Ni-column, forming complexes by the co-transfection and followed pull-down, because in the Western blot the 3 interest proteins could be recognized by specific antibody in the co-transfected products, but only B-LB-Sβ1 and B-LB-β1 were found as the complexes (His/Ii and B-LB-Sβ1, His/Ii and B-LB-β1), which were dissociated from Ni-column in pull-down. Finally it was proved that B-LB-Sβ1 and B-LB-β1 could express and localize in the endometrial system of 293T cells, which kept the function such as whole MHC molecule, but the B-LB-β2TC lost this function. All of above results first time provide an evidence of binding of active segments of MHC class Ⅱmolecules with Ii in vitro.
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Received: 16 May 2016
Published: 06 August 2016
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