Binding of Grass Carp (Ctenopharyngodon idellus) Invariant Chain (Ii) with Its Major Histocompatibility Complex (MHC) Molecules Detected with a Pull-down Method
Abstract：Invariant chain (Ii) can bind to major histocompatibility complex (MHC) classⅠorⅡmolecules, and plays an important assistant role in antigen presentation by MHC molecules. In order to explore what about the binding of Ii with MHC molecules, a Pull-down method for detection of association of grass carp(Ctenopharyngodon idellus) Ii with MHC molecules was reported in the present study. DNA segments of Ii (711 bp), MhcⅠα (540 bp) and MhcⅡβ (549 bp) in grass carp were cloned, and then respectively inserted into prokaryotic expression plasmid PET-22b (no His-tag) or PET-28a (carry with His-tag) to construct 3 recombinant plasmids of PET-22b-Ii, PET-28a-MhcⅠα and PET-28a-MhcⅡβ. The above 3 recombinant plasmids then were transfected into engineering bacteria (Escherichia coli) Rosetta (DE3), respectively. After an induction to express and renature the interest proteins and purification by Ni+-NTA affinity chromatography column, the purified expressed protein products were detected by polyacrylamide gel electrophoresis (PAGE). The results indicated that MHCⅠα and MHCⅡβ labeled with His-tag could well express and be purified each with a molecular weight of 24 kD. The result of Western blot showed that Ii also well expressed and had a molecular weight of about 30 kD. Finally, the recombinant plasmid PET-22b-Ii was cotransfected with PET-28a-MhcⅠα or PET-28a-MhcⅡβ into Rosetta (DE3), respectively, to construct 2 recombinant strains of Rosetta(DE3)(PET-28a-MhcⅠα+PET-22b-Ii) and Rosetta(DE3)(PET-28a-MhcⅡβ+PET-22b-Ii). Induced expression and PAGE detection showed that MHCⅠα or MHCⅡβ could bind Ii to form a complex of 54 kD, respectively, which could be dissociated into simple molecule of Ii (30 kD), MHCⅠα or MHCⅡβ (24 kD) after SDS treatment, respectively. Western blot result showed that the dissociated Ii could bind specific antibody and be coloured by electrogenerated chemiluminescence (ECL). In conclusion, the prokaryotic expressed grass carp Ii and MHC molecules had activity, and its Ii could bind with MHCⅡβ as well as MHCⅠα. This research provides basic data for further research on relation between Ii and MHC molecules in animals.
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