Construction Of Gene Of Single Chain Variable Fragment Against Acetylcholine Receptor In Myasthenia Gravis

Antibody competition experiments suggest that about 2/3 of the antibodies in the sera of myasthenia gravis (MG) patients are directed against the main immunogenic region (MIR) of acetylcholine receptor (AChR). Univalent antibody fragments, such as single chain variable fragment (ScFv), do not cross-link AChR molecules or bind complement, and therefore do not cause AChR loss; on the contrary, such fragments derived from anti-MIR monoclonal antibodies (mAbs) seem capable of protecting the receptor against the loss induced by intact anti-MIR mAb or MG sera.The heavy chain variable region (VH) gene of a mAb against AChR amplified by polymerase chain reaction (PCR) was purified by Wizard PCR Preps DNA Purification System, then digested with NcoⅠand XhoⅠ. The digested products were run in low gelling temperature agarose eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into plasmid pHEN2 digested and purified by the same method. There recombinant pHEN2-VH were transformed into E.coli DH5αfor amplification and isolated from E.coli DH5αand digested with NcoⅠand XhoⅠagain. The light chain variable region (VL) gene of the mAb was cloned to pHEN2-VH by the same ways and than the recombinant plasmids were transformed into E.coli HB2151. The VL gene was analysed for an insert of the right size by digestion with ApalⅠand NotⅠ. The sequencing showed that the nucleotide sequence of cons-tructed ScFv was correct and cloned into the open reading frame (ORF) in pHEN2. A ScFv gene against the MIR of AChR has been successfully constructed.