Dystrophin gene is the largest in the human genome with 79 exons covering greater than 0.1% of the total genome, located on the Xp21 locus of... Show moreDystrophin gene is the largest in the human genome with 79 exons covering greater than 0.1% of the total genome, located on the Xp21 locus of the “X” chromosome resulting in a 427kDa protein, “Dystrophin”. Dystrophin is an important cytoskeletal protein which belongs to the β-Spectrin/α-actinin family of proteins. It comprises of an amino terminal domain, alpha helical coiled structure COOH domain, central rod region with 24 STRs and four proline rich hinge regions. It plays a vital role in localizing the Dystrophin glycoprotein complex (DGC) in the Sarcollema and is associated with the DGC in controlling the signaling events of certain proteins associated with DGC. The large size of the gene makes it more vulnerable to mutations resulting in partially functional or non-functional Dystrophin. The absence of Dystrophin results in disruption of sub sarcolemma-extracellular matrix linkage, loss of nitric oxide, progressive muscle weakening and muscle wasting leading to the death of patients typically before the end of their teenage. In certain cases alternatively spliced isoforms produce Dystrophin with reduced length yet stable and completely functionality. The main focus of this project was to select monospecific antibody against the more stable alternatively spliced variant D14 (15”16”) 17, which is functional and more stable compared to unspliced parent D14:17. The yeast surface display technique was used to effectively screen and select the yeast scFv clones containing the monospecific antibody against our target spliced variant D14 (15”16”) 17 protein and D2:3. The yeast scFv sub population was enriched by repeated MACS and FACS selection. Test colonies picked from the enriched scFv pool were confirmed via PCR and restriction digestion analysis. The scFv for the respective antigens were then sub cloned into pPnnl-9 secretion vector using YVH10 yeast cells via LiTRAFCO method. It was clear that by repeated MACS and FACS selection the scFv pool can be enriched and the yeast scFv clone sub population can be reduced to a significant level. The scFv sub cloned into Pnnl-9 secretion vector can be purified using affinity chromatography and the further affinity and avidity studies can be conducted. M.S. in Biology, May 2011 Show less
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(-) mods_name_creator_namePart_mt:"Saraswathi, Raj Prabu Vijayakumar"
