title: Potential roles of recombinant acetylcholine receptor α subunit 1-211 in immunoadsorbent and DNA immunization.
abstract: The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) 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 hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). 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 hAChR(α211) immunization.
authors.affiliation: Institute of Biotechnology, Laboratory of Chemical Biology and Molecular Engineering of National Education Ministry, Shanxi University, Taiyuan 030006, PR China.
authors.forename: Lixi
authors.initials: L
authors.lastname: Niu
authors.forename: Chenyun
authors.initials: C
authors.lastname: Guo
authors.forename: Zhibo
authors.initials: Z
authors.lastname: Hao
authors.forename: Jingming
authors.initials: J
authors.lastname: Yuan
authors.forename: Zhuoyu
authors.initials: Z
authors.lastname: Li
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D006801:Humans; D007164:Immunosorbents / Q000494:pharmacology* / Q000627:therapeutic use; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009157:Myasthenia Gravis / Q000276:immunology*; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology
chemical_list: D007164:Immunosorbents; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA