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PROTEIN EXPRESSION AND CHARACTERIZATION OF THE MAJOR AUTOANTIGEN (TITIN DOMAIN) ASSOCIATED WITH AUTOIMMUNE RIPPLING MUSCLE DISEASE

Zelinka, Lisa M
http://rave.ohiolink.edu/etdc/view?acc_num=kent1416852571

Abstract Details

2015, PHD, Kent State University, College of Arts and Sciences / School of Biomedical Sciences.
An autoimmune disease is distinguished by the appearance of several autoantibodies. These autoantibodies can react with components of surface, cytoplasmic or nuclear origin. Autoantibodies can confirm diagnosis and prognosis as well as monitor progression of a certain autoimmune disease. Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Rippling muscle disease is diagnosed by percussion or stretch stimulated wave-like muscle contractions. The propagation of these contractions does not involve motor unit action potentials (muap). Past experiments in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform N2-A, ATP synthase 6 and PPP1R3. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. Previous experiments in our laboratory have subcloned the immunoreactive domain of titin isoform N2-A into pGEX-3X G-S-T fusion vector (G3RMMG6). Sequence analysis of the glutathione-S-transferase/Titin N2-A fusion gene indicates the cloned titin domain (GenBank accession # EU428487) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression and affinity purification of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, shows that only the titin domain is immunoreactive. Current experiments in our laboratory have affinity purified the autoantibody from the serum of an (ARMD) (MG) and thymoma patient using the Olmsted method. The affinity purified autoantibody was used for immunofluorescent microscopy. The Olmsted affinity purified autoantibody demonstrated immunoreactivity in a clear and concise striational banding pattern consistent with the striational banding pattern of the I and A bands of human skeletal muscle. Future studies will use the tools developed in this study to explore the functional role of the exon 248-250 domain in muscle contractility.
Gary Walker, Dr. (Committee Chair)
Eric Mintz, Dr. (Committee Member)
Fayez Safadi, Dr. (Committee Member)
Wen-Hai Chou, Dr. (Committee Member)
Robert Clements, Dr. (Committee Member)
150 p.
Bioinformatics; Biomedical Research
Autoimmune Rippling Muscle Disease; Myasthenia Gravis; Autoantibody; Protein expression; Protein Purification; Genetic Manipulation of E coli; Bioinformatics

Recommended Citations

Citations

  • Zelinka, L. M. (2015). PROTEIN EXPRESSION AND CHARACTERIZATION OF THE MAJOR AUTOANTIGEN (TITIN DOMAIN) ASSOCIATED WITH AUTOIMMUNE RIPPLING MUSCLE DISEASE [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1416852571

    APA Style (7th edition)

  • Zelinka, Lisa. PROTEIN EXPRESSION AND CHARACTERIZATION OF THE MAJOR AUTOANTIGEN (TITIN DOMAIN) ASSOCIATED WITH AUTOIMMUNE RIPPLING MUSCLE DISEASE . 2015. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1416852571.

    MLA Style (8th edition)

  • Zelinka, Lisa. "PROTEIN EXPRESSION AND CHARACTERIZATION OF THE MAJOR AUTOANTIGEN (TITIN DOMAIN) ASSOCIATED WITH AUTOIMMUNE RIPPLING MUSCLE DISEASE ." Doctoral dissertation, Kent State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=kent1416852571

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Kent State University and OhioLINK.