The Preparation And Functional Test For Tetrameric SH-2K～d-HBc Complexes

Objective In order to research the mechanisms of specific CLT responding and effecting and to explore the methods of breaking through the immune tolerance to some extent in chronic hepatitis B patients, we construct the tetrameric sH-2Kd-HBc complex which can offer a powerful way for detecting and separating peptide-specific T cells.Methods The extracellular region H-2Kd were amplified from the clone plasmid temple by PCR., and a 15-amino acid substrate peptide for BirA-dependent biotinylation was added to the COOH-terminus of H-2Kd heavy chain. The products were subcloned into pET-28(a+) vector. The reconstructed vectors pET-sH-2Kd-BSP and pET-β2M which was previously constructed by our lab were transformed into E. coli strain BL21(DE3)plysS respectively and overexpressed. After purified, the fusion proteins were refolded into sH-2Kd-HBc complex in the presence of an H-2Kd-restricted antigenic peptide by dilution method. The complex then was biotinylated by BirA enzyme and purified via the gel-filtration chromatography, Here we use HRP-streptavidin to detect the biotinylated product. Finally the corresponding tetramer was prepared by mixing the biotinylated complex with phycoerythrin(PE)labled avidin and apply the prepared tetramer to stain the specific T cells which were induced by genetic immunization to the mice.Results1,Direct sequencing results showed that the homologies of nucleotides and amino acids between presumed and the sequence we got were 100%. The proteins were analyzed by SDS-PAGE and Western blot, and were found in E. coli as insoluble aggregates . We harvested 186mg sH-2Kd-BSPand 165mgβ2M with 500ml saturant bacterial suspension, and the purity of the two proteins were both 70%, respectively.2,We use HRP-streptavidin to detect the biotinylated product. The result demonstrates the refolded complex has been biotinylated successfully.3,We apply the prepared tetramer to stain the specific T cells which were induced by genetic immunization to the mice. The result shows our tetramer can discriminate the frequencies of specific CTL induced by immunization. The techniques and methods can be used for preparation of tetramers of other types of MHC-I molecules and evaluation of distinct immune effects initiated by different vaccination methods.Conclusion1,The expression vectors of recombinant sH-2Kd-BSP fusion gene were constructed successfully. sH-2Kd-BSP fusion gene andβ2-microglobulin(β2m) gene can express in prokaryotic system, and Both the quality and the quantity of the proteins can afford to the preparation of sH-2Kd-HBc complex. 2,The results indicated the success of refolding performed by dilution using a appropriate molarratio of heavy chain :light chain: peptide. The complex then was biotinylated by BirA enzyme and purified via the gel-filtration chromatography to get rid of the unrefolding heavy chain and light chain from refolding system to purify the biotinylated complex furtherly. 3,We can induce the strong specific T cells response by genetic immunization to the mice. Our prepared tetramer can bind to the target cells specifically, so as to provide the basis for the further studying the mechanisms of T cells recognition.Significance1,As the ligands of antigen-specific T lymphocytes, H-2Kd -peptide complexes can be applied to the preparation of artificial antigen-presenting cell (artificial APC) which offers a new way for increasing or decreasing the amount and function of antigen-specific T cells. The corresponding tetramer can quantify and isolate the specific T cells for the further studying the mechanisms of T cells recognition.2,The techniques and methods we groped can also be used for preparation of other types of MHC class I molecules and the corresponding tetramer. For this reason, our research offers experimental basis to reveal the mechanism of virus infection, tumors occurrence, transplant rejection and autoimmune diseases, and helps to search for the corresponding immunotherapies.