Construction And Expression Of The Fusion Gene Of Anti-acetylcholine Receptor Single Chain Variable Fragment And Human Serum Albumin In Myasthenia Gravis

Objective To increase the stability of single chain variable fragment against the main immunogenic region in human acetylcholine receptor by preparing the fusion protein of single chain variable fragment and human serum albumin. Methods: Human serum albumin gene was amplified by PCR and cloned in vector pHEN2 containing single chain variable fragment against human acetylcholine receptor (pHEN2-ScFv637). The recombinant plasmid pHEN2-ScFv637-HSA gene was subsequently transformed into E coli DH5 a . After isolation and purification, the recombinant was digested with endonucleases and checked in agarose gel electrophoresis to confirm the right size of the human serum albumin gene and the fusion gene. The fusion gene was sequenced after checked in agarose gel electrophoresis. The fusion gene in the vector pHEN2 bearing a pelB leader and a c-myc tag were expressed in periplasm of E coli HB2151. The expression was detected by dot blotting assay using a mouse anti-c-myc monoclonal antibody. Molecular weight of fusion protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Results: The sizes of the human serum albumin gene and fusion gene were found to be approximately 1770 bp and 7054 bp respectively, and the inserted positions were correct. The sequencing showed that the nucleotide sequence of constructed ScFv637-HSA gene was correct and cloned into the open reading frame (ORF) in pHEN2. Fusion protein containing single chain variable fragment and human serum albumin was found in periplasmic fraction, however no expression was observed in culture medium, or in periplasmic fraction and culture medium of control E coli HB2151. Molecular weight of fusion protein was found to be approximately 111.4 KD by SDS-PAGE and western blotting, whereas no expression was detected in periplasm of control E coli HB2151.Conclusion: Recombined fusion protein has been prepared successfully. These lay a foundation for the further research on its application.