The Optimization Of Expression And Purification Of The Soluble I-A~b/Fc Dimer

Major histocompatibility complex (MHC) is a group of closely linked with ahighly polymorphic gene cluster in the vertebrate of chromo some. MHC geneproducts can be divided into two types, depending on the structure and function ofMHC Ⅰ and MHC II molecules.The MHC II molecules is rendered in the surface ofspecific cells of the immune system by the A and B chains of non-covalently bondedto the group consisting of highly polymorphic transmembrane glycoproteins[1].Recently it was found that soluble MHC II molecules (especially in the form ofpoly-soluble pMHC) and epitope-specific T cell binding, can play the role in immuneregulation. The mechanism is as follow:①It closes specific TCR;②solublePMHC combined with TCR can only provide the T cells activated by the first signal,but the lack of a second signal will cause T cell deactivation and clonal deletion,which can suppress T cell responses to antigen.Early in our laboratory, our lab causedC57BL/6mouse occurred EAE by usingantigen peptide MOG35-55, and t cell epitopes which are relatively clearcan induceautoreactive CD4+T cell responses, causing immunologic injury. The laboratory hasbeen constructed several soluble pMHC (MOG35-55-I-Ab/Fc) dimerswhich isspecifically bound to CD4+T cells. The pMHC dimer acts on the EAE mice model invivo intervention experiment, in order to be able to alleviate the symptoms of mouse EAE model.However, there were some problems that the expression and biologicalactivity were not good in several I-Ab/Fc dimerprepared previously, and we couldn’tpurify the I-Ab/Fc effectively very much because we just concentrated dimer withPEG incompletely, so in this way, it was very unfavorable to explore the therapeuticeffect and mechanism of EAE for further experiment. We should increase the outputof the expression of dimer andto optimize the purification conditions in order to meetour target. So The main contents and results of the experimental study are as follows:1the eukaryotic expression vector containing insect signal peptide genesequencewas successfully constructedUsing the signal peptide to guide the secretion of exogenous protein localizationto the cell-specific interval to increase soluble can be avoided the difficulties broughtby due to inclusion bodiesrefoldingy[2].Prokaryotic expression system, such as lacticacid bacteria, E. coli,etc.has been expressed and secreted, signal peptide is also widelyused in the eukaryotic expression systems such as the Pichia yea st and baculovirusexpression system[3].In order to improve the level of I-Abdimer protein secretion, weuse the Primers connected withinsect signal peptide to plasmid which was constructedby our lab beforeby overlapped PCR technology,enzyme digestion andconnectiondigested and connected,then formed a objective fragement with insectsigal pepite. We would load I-Ab-a-Fos and IgGFcγ2b to the downstream of the PHand P10,thus we would get the pFastBacTMDual[I-Ab/Fc] recombinantplasmid.Therecombinant gene was sequenced to guarantee that no nonsense mutationwere introduced during the PCR and the result shows that eukaryotic expressionvector is successfully constructed.2The function, the optimization of expression and purification of the solubleI-Ab/Fc moleculeAccordingto the bac-to-bac insect expression system instructions,we transtecedthe constructed plasmids,such as K-MOG，K-MOG without MOG peptide,MOG,andI-Ab/Fc without MOG peptide into SF9cell respectively,then extractedbacmidseverally. Next step,we transferred the fourbacmids intoSF9cell and extracted cell supernatantsin synchronization with extracted gp67-I-Ab/Fc dimer，andthen tested by western blotting and elisa.Finally，we found that the level of proteinexpression with kozak sequence was the highest in these dimers,it also had a goodconformation andfunction,so the result indicated the level of K-MOG expression hadachieved our expection.What next to do was to purify Kozak-MOG well to lay agood foundation for further experiments. Finally,the purified I-Ab/Fc (K-MOGdimer) dimereffectsEAE mouse T cells in vitro,andinhibits the T cell proliferativeresponsesuccessfully.The K-MOG dimer iseffective, and it shows that the screeningprocess and the purification process isright,have reached expected results.