Potential roles of recombinant acetylcholine receptor α subunit 1–211 in immunoadsorbent and DNA immunization

The open reading frame of the α-subunit (amino acids 1–211) of human muscle nicotinic acetylcholine receptor (hAChRα211) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChRα211 (recombinant hAChRα211) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K–hAChRα211 with a yield of 25 mg/L. rhAChRα211 was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChRα211 was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA–hAChRα211. Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChRα211 immunization.

► Using yeast expression system, recombinant human AChRα211 (rhAChRα211) was obtained.
► Immunoadsorbent prepared by coupling the rhAChRα211 with CNBr-actived Sepharose 4B.
► By this immunoadsorbent, AChRAb was efficiently removed from the patient's sera.
► Mouse model of myasthenia gravis was established by DNA immunization.