Construction Of MuSK-mCherry Fusion Fluorescent Protein And Gene Polymorphisms Of Rapsyn In Myasthenia Gravis

Construction of MuSK-mCherry fusion fluorescent protein and used for the detection of MuSK antibodies in myasthenia gravisObjective:To construct a fusion protein of extracellular domain peptide fragment of muscle specific kinase (MuSK) and fluorescent protein mCherry, and used as antigen in the detection of antibodies against MuSK (MuSKAb) in the sera of patients with myasthenia gravis (MG). Methods: The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector, and furthermore, cloned into Eukaryotic expression vector pMT/BiP/V5-His(MuSK), which contains MuSK extracellular domain 22-452 amino acid peptide fragment gene to construct the fluorescent fusion protein gene MuSK-mCherry. The recombinant vector was subsequently transfected into drosophila S2 cells for expression. The expressed fusion proteins were verified in confocal microscope, and used as antigen in the detection of MuSKAb in sera of MG patents in fluorescence immunoprecipitation test. Results:The fluorescent fusion protein MuSK-mCherry was successfully constructed and expressed. The MuSKAb in sera of patents with MG could be detected in fluorescence immunoprecipitation test using the constructed MuSK-mCherry fusion protein as antigen. Conclusion:It is available to use the constructed fluorescent fusion protein MuSK-mCherry as antigen in fluorescence immunoprecipitation test for the detection of MuSKAb in sera of patents with MG.Association of gene polymorphisms of rapsyn exons with myasthenia gravisObjective:To investigate the association of single nucleotide polymorphisms (SNPs) of receptor-associated protein at the synapse (rapsyn) with myasthenia gravis (MG.). Methods:The genomic DNA was extracted from peripheral blood cells, sampled from 132 patients with MG and 153 control individuals. The 8 exons of rapsyn gene were amplified by PCR, then the products of PCR sequenced directly. Each sequence was compared with wild-type rapsyn gene, and the association between mutation and clinical symptoms of MG analysed. Results:No mutation was found in the exons 1,2,4,5,6,7, and 8 of rapsyn gene both in MG patients and control group compared with the wild-type rapsyn gene. However, a new SNP, L222R[CTG>CGG(2)] or T665G, was found in exon 3. The allele and genotype frequencies of SNP L222R were in accordance with Hardy-Weinberg genetic equilibrium (P>0.05), indicating the group representativeness. The allele frequencies of G were not statistically different between patient and control groups (P> 0.05). There were differences in the 3 genotypes TT, TG and GG between patient (42.4%V48.5%V9.1%) and control (49.0%V33.3%V17.6) groups (P<0.05). The genotype frequencies of GG were statistically higher in control group than that in patient group, showing a recessive model of inheritance. Conclusion:The SNPs in the rapsyn gene are associated with MG in this study. L222R(T665G) is a new SNP found and allele G might be a protective factor for MG.