Application Of Eukaryotic Expression Of Agrin In The Experimental Diagnosis Of Myasthenia Gravis

Background and PurposeAgrin is an extracellular matrix protein synthesized and released by the terminal of motor neurons.The human AGRN gene is located on chromosome1p36.33.The molecular weight of Agrin protein is 217 k Da and there are 6transcripts and 5 protein variants.The AGRN gene is divided into two tissue-specific subtypes through different shearing modes: muscle-type agrin(M-agrin)and neural-type agrin(N-agrin).M-agrin is expressed in skeletal muscle and other tissues.M-agrin lacks the insertion of 8 specific amino acids at the Z insertion site.Therefore,the ACh R clustering activity of M-agrin is at least 1000 times lower than the ACh R clustering activity of N-agrin.N-agrin is synthesized and secreted by the terminal of motor neurons.It is also a regulator of the interaction between the transmembrane protein low-density lipoprotein receptor-related protein 4(Low-density lipoprotein receptor-related protein 4,LRP4)and muscle-specific receptor tyrosine kinase(Muscle-specific receptor tyrosine kinase,Mu SK).N-agrin plays an important role in the formation,maintenance and regeneration of neuromuscular junction(Neuromuscular junction,NMJ).Myasthenia Gravis(Myasthenia gravis,MG)is an autoimmune disease characterized by partial or systemic skeletal muscle weakness and fatigue.N-agrin released from the terminal of motor neurons binds to LRP4,activates Mu SK to make it phosphorylate,and induces the accumulation and expression of nicotinic acetylcholine receptor(nicotine Acetylcholine receptor,n ACh R)on the muscle cell membrane,allowing the normal transmission of excitement.The presence of Agrin antibody(Agrin-Ab)can lead to neuromuscular transmission disorders,thereby causing the occurrence of MG.As a new type of MG pathogenic antibody,the clinical features and prognosis of Agrin-MG patients are still unclear.So far,different scholars have established non-full-length Agrin based CBA and ELISA detection systems worldwide,and only a few cases have been detected.There is no report on Agrin-MG patients in China.In this study,a eukaryotic expression plasmid of Agrin was constructed based on human full-length Agrin and transfected into HEK293 T cells to establish a method for the detection of Agrin-Ab by cellular immunofluorescence,to detect Agrin-Ab in the serum of Chinese MG patients,and to explore the clinical practice of Chinese Agrin-MG patients.Methods1.Query the human full-length AGRN gene sequence in the NCBI database,design specific primers according to the full-length c DNA fragment of human Agrin transcript 1 encoding protein,and use overlap shear extension PCR technology to amplify Agrin from human brain tissue Gene,connect it to the empty p CMV6-AC-GFP vector and named it p CMV6-AC-Agrin-GFP.2.Transform and optimize the constructed Agrin plasmid,and re-construct four eukaryotic expression Agrin plasmids: pc DNA3.4-Agrin-GFP,pc DNA3.4-GFP-Agrin,p CAGGS-2-Agrin-GFP,p CMV6-AN-Agrin-GFP.Five different Agrin plasmids were transfected into HEK293 T cells,and the fluorescence intensity and transfection efficiency were compared.3.The p CMV6-AC-Agrin-GFP was subjected to single and double enzyme digestion and sequencing verification.The constructed p CMV6-AC-Agrin-GFP plasmid was transfected into HEK293 T cells,and the full-length Agrin protein expressed by the cells was verified by Western blot and immunofluorescence.4.Collect clinical data and serum samples of patients diagnosed with MG.Use enzyme-linked immunosorbent assay to detect ACh R-Ab and Titin-Ab,and use cellular immunofluorescence method to detect Mu SK-Ab and LRP4-Ab.5.Establish a cellular immunofluorescence detection method for Agrin-Ab,and perform Agrin-Ab detection on collected patient serum.6.Use IBM SPSS to perform statistical analysis on the overall information of MG patients,and summarize the demographic and clinical characteristics of Agrin-MG.Results1.Construct five eukaryotic expression plasmids of Agrin,and finally select the p CMV6-AC-Agrin-GFP plasmid,and use single and double enzyme digestion and sequencing to identify the plasmids,confirming the successful construction of the plasmids.2.Compare the fluorescence of five different eukaryotic expression Agrin plasmids,and finally select the p CMV6-AC-Agrin-GFP plasmid with the GFP tag at the carboxyl end of Agrin.3.The p CMV6-AC-Agrin-GFP plasmid was transfected into HEK293 T cells to express human full-length Agrin protein.Western blot and cell immunofluorescence methods proved the successful expression of the protein.4.Uellular immunofluorescence was used to detect Agrin-Ab and related antibodies in the serum of Chinese MG patients.Among the 1948 confirmed MG patients,the antibody frequency was: Agrin-Ab,0.92%;ACh R-Ab,71.66%;Mu SK-Ab,2.56%;LRP4-Ab,0.78%;Titin-Ab 17.9%.Among triple-negative patients,the positive rate of Agrin-Ab was 1.82%.5.Among the 18 patients with Agrin-MG detected,9 patients were Agrin-Ab alone and 9 patients were combined with ACh R-Ab(2 of which were combined with thymoma).The male to female ratio of the 18 patients was 1:0.64,Among them,11 were male patients,and the age of onset was all older than 40 years,the proportion was 61.11%;there were 7 female patients,and the age of onset was less than 1 year old or older than 35,the proportion was 38.89%.6.The first symptom of Agrin-MG is mostly ocular muscle weakness,the proportion is 81.25%(13/16).The remaining patients have limb girdle muscle or bulbar muscle as the first symptom.7.The clinical symptoms of Agrin-MG are usually severe,and the effect of treatment with brompistigmine or prednisone alone is not significantly improved,and the patients’ symptoms can be effectively relieved when the two drugs are treated in combination.Conclusions1.The human full-length Agrin plasmid was successfully constructed and correctly expressed.Cellular immunofluorescence can detect Agrin-Ab in the serum of Chinese MG patients.2.The positive rate of Agrin-Ab in China is low,with a total positive rate of0.92%;the positive rate of Agrin antibody in triple-negative serum is 1.82%.3.Agrin-MG have unique clinical characteristics,including: more common in middle-aged and elderly men;more ocular muscles are involved,but also bulbar muscles and limb muscles;may be combined with ACh R-Ab positive,and may be combined with thymoma;clinical symptoms are usually It is heavier,and has a better effect on the combined treatment of brompistigmine and prednisone.