Construction Of Eukaryotic Expression Vector Of A7 Gene Of Single Chain Variable Fragment Against Acetylcholine Receptor In Myasthenia Gravis

Myasthenia gravis (MG) is an organ-specific autoimmune disease mediated by autoantibodies directed against acetylcholine receptor (AchR). AchR is a transmembrane glycoprotein, consisting of five subunits α₂βγδ. The antibodies in sera of MG patients are mainly those direct against the main immunogenic region (MIR) on a-subunit of AchR. The damage of postsynaptic membrane and AchR loss by antigen modulation and activation of complement result in muscle weakness, when pathogenic antibody bind to MIR of consecutive two a-subunit of AchR on postsynaptic membrane in neuromuscular junction. As univalent antibody fragments, single chain variable fragment (ScFv) which derived from antibody against AchR can bind to only one MIR of a-subunit, therefore it can not cause antigen modulation. It also can not cause the damage of AchR by activation complement because of having no Fc fragment. However such fragments are capable of protecting the receptor against the loss induced by intact anti-MIR andibodies.In this experiment the mouse ScFvA7 gene amplified by PCR from the recombinant vectors pHEN2-ScFvA7 was purified by Wizard PCR Preps DNA Purification System, then digested with EcoR I and Avr â…¡. The digested products were run in low melting point agarose gel eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into eukaryotic expression vectors pPIC9K digested and purified by the same method. The recombinant vectors pPIC9K-ScFvA7 were transformed into E. coli DH5a for amplification and isolated from E. coli DH5a and digested with...