Expression Of A Cetylcholine Receptor Tetramer Precursor A Subunit On The HEK293 Cells Membrane

Objective:To express the recombinant vector bearing acetylcholine receptor (AchR) a subunit (pcDNA3.1-AchRa-BirA) on the membrane of human embryonic kidney 293 (HEK293) cell line as first step toward the construction of AchR tetramers, to be used as an antigen in determination of anti-AchR antibodies (AchRAb) in radioimmunoassay (RIA).Methods:The recombinant plasmid vector pcDNA3.1-AchRa-BirA was constructed using molecular cloning technology, and transformed into E. coli DH5a for amplification. The recombinant plasmid vector was obtained from the E. coli DH5a, and then analyzed by low-melting agarose gel electrophoresis and the gene sequencing. Furthermore the recombinant plasmid pcDNA3.1-AchRa-BirA was transfected into HEK 293 cell by the liposome-mediated method. The clones with high expression were selected by antibiotics G418. The gene expression of pcDNA3.1-AchRa-BirA on the membrane of HEK 293 cells was analyzed by immunofluorescence technology.Results:A 6964 bp target fragment was observed in agarose gel electrophoresis, and the sequencing also showed that the plasmid sequences were correct. After transfection, several expression clones were obtained with the selection of G418. The green fluorescence on the membrane of HEK293 was observed by fluorescence microscopyConclusions:The recombinant vector pcDNA3.1-AchRa-BirA recognition polypeptide sequence gene was successfully extracted, and expressed on the membrane of HEK293 cells.