The Application Of Eukaryotic Expression Of HMGCR In The Experimental Diagnosis Of Immune-mediated Necrotizing Myositis

ObjectiveAnti-3-hydroxy-3-methylglutaryl-Co A reductase（HMGCR）antibody is a specific antibody of immune necrotizing myopathy and a newly discovered immunological marker in recent years.The purpose of this study is to construct three different eukaryotic expression vectors pSJMY-H-mcherry,pSJMY-H-GFP_Cand pSJMY-GFP_N-H of HMGCR with fluorescent tags,these three vectors are vectors expressed in eukaryotic cells.The method was constructed by liposome transfection,and the anti-HMGCR antibodies in the serum of the patients was determined.Methods1.Detection of HMGCR gene by RT-PCRHuman muscle tissue was obtained from clinical practice and total RNA was extracted and total c DNA,was obtained from NCBI to obtain HMGCR sequence.We chose to separate the HMGCR gene into two segments for amplification,the front segment and the back segment,designed appropriate upstream and downstream primers,and used the designed and synthesized primers for RT-PCR to obtain human HMGCR gene.2.Construction and Identification of Recombinant expression VectorIn this study,we selected two kinds of fluorescent tags to construct the vector of two-color indirect immunofluorescence.They are GFP green fluorescent protein and mcherry red coral protein.We constructed an empty vector containing fluorescent tags,pSJMY-GFP and pSJMY-mcherry.At the same time,the fluorescent vector was identified by PCR,restriction enzyme digestion and gene sequencing.The recombinant anterior and posterior fragments were inserted into the empty vectors pSJMY-GFP_C,pSJMY-GFP_N,and pSJMY-mcherry,and three-fragment ligation or direct seamless cloning was done,and positive clones were selected after transformation.The recombinant vectors pSJMY-H-mcherry,pSJMY-H-GFP_Cand pSJMY-GFP_N-H were constructed,and then the recombinant plasmids were identified by target gene PCR and restriction enzyme digestion.Selected the correct vector for a large number of plasmid extraction,sterilization and other work.3.Establishment and verification of CBA（Cell-Based Assay）indirect immunofluorescence methodThe recombinant plasmids of pSJMY-H-mcherry,pSJMY-H-GFP_Cand pSJMY-GFP_N-H were transfected into human 293T cells.Under the condition of high transfection efficiency,the transfected cells were treated by fixation,membrane breaking,preparation and other steps.Through HMGCR primary antibody,the experimental conditions of two-color indirect immunofluorescence method were explored and the method was finally established.The expression of the target protein in the cells was verified by Western Blot experiments.4.Application in the screening of HMGCR antibodies in clinical serum samplesPeripheral blood samples were collected from several patients with myasthenia gravis（MG）,and the anti-HMGCR antibodies in human serum was detected by two-color indirect immunofluorescence method.And the results of anti-HMGCR antibodies were derived according to the order of excitation light observation of two different types of fluorescent labeled carriers,and the significance of CBA method for detecting anti-HMGCR antibodies in the diagnosis of immune necrotizing myopathy was analyzed and summarized.Result1.Three vectors of pSJMY-H-mcherry,pSJMY-H-GFP_Cand pSJMY-GFP_N-H with correct size and position were constructed,and the recombinant plasmids were identified by PCR,restriction endonuclease digestion and gene sequencing.2.WB verification results showed that the fusion protein band appeared around120 k D.3.The CBA two-color indirect immunofluorescence method was established,used the two-color indirect immunofluorescence method to determine the anti-HMGCR antibodies in human serum,and selected 6 positive samples from 149myositis serum samples,the positive rate is 4%.Conclusions1.Successfully constructed three HMGCR eukaryotic expression vectors:pSJMY-H-GFP_C,pSJMY-GFP_N-H,pSJMY-H-mcherry.2.A two-color indirect immunofluorescence method based on 293T cells was established.3.The two-color indirect immunofluorescence method can be used to determine the anti-HMGCR antibodies in the serum of the patients,and the double-color coincidence was determined to be positive,which significantly improved the specificity and sensitivity of antibody detection.