Construction, Expression And Identification Of Divalent MOG35-55-I-A~b/FC Molecule With MOG35-55 Covalently Attached

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating CNS disorder that serves as a model for the human multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is a key autoantigen involved in the development of demyelinating lesions in EAE and induces not only an encephalitogenic T-cell response in susceptible species but also a demyelinating autoantibody response. It is clear that peptide MOG35-55 can induce EAE in C57BL/6 by activate the autoreactivity CD4+ T cells. Soluble peptide/major histocompitibility complex class II (pMHC II) dimer, which is prepared by a peptide covalently attached to the N terminal of chain of MHC class II molecule, and Fc region of IgG linked to the C terminal of MHC II a chain, can be used to detect specific CD4+T cells and modulate the immune response by interaction with T cell receptors (TCR). The dimer is usually expressed in eukaryotic cells because of the complicated structure. Baculovirus expression system is now widely used for the expression of dimeric MHC class II molecule. In this study, we constructed a recombinant expression vector of pFastBac Dual+[MOG35-55-I-A /Fc], which consists of the extracellular region of I-A~bDNA and IgG2b Fc region DNA. The peptide MOG35-55 is covalently attached to the N terminal of B chain and a leucine zipper is fused at the C terminal. The fusion molecue is expressed in the baculovirus expression system. This work provides a tool for the studies of the mechanism of EAE and the therapy of autoimmune disease.The work contains the following two major parts:1. Construction of pFastBacTMDual+[MOG35-55-I-A~b/Fc] plasmidThe MOG-I-A -Jun fusion gene was consturcted by covalently attachingMOG35-55 to the N terminal and the Jun gene to the C terminal of the extracellular domains of I-A . I-A -Fe Fos was constructed by fusing the extracellular domains of I-A chain with the Fc portion of IgG2b and Fos gene at the C terminal. The two fusion genes were cloned into the double expression plasmid pFastBac Dual at the down stream of protmotor PH and P10 respectively to form pFastBac?Dual+[MOG35-55-I-A~b/Fc] plasmid. The entire fusion gene was sequenced to ensure that no spurious mutations were introduced during the PCR. The results show that the recombined double expression plasmid was constructed.2. Expression and identification of MOG35-55-I-A /Fc fusion proteinThe recombined double expression plasmid pFastBac Dual+[MOG35-55-I-A /Fc] was transformed into host bacterium DH10 E coli, and formed a recombined baculovirus bacmid containing the interest genes of I-A~b -Fe Fos and MOG-I-A~b -Jun. The recombined baculovirus bacmid was then transfected into the insect cell line sf9 with liposome Cellfection2000. The supernatant of the infected cells contained the fusion protein and the baculovirus virion which could infect the normal insect cell sf9 for further protein expression. Sandwich ELISA, SDS-PAGE and flow cytometry with specific antibody suggested that the fusion protein MOG35-55-I-A /Fc was expressed in the sf9 cells with expected molecular weight and intact I-A comformation.