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Generation Of Monoclonal Antibody Specific Against α-Enolase And Dissect The Charactization

Posted on:2016-05-28Degree:MasterType:Thesis
Country:ChinaCandidate:D HanFull Text:PDF
GTID:2180330464471891Subject:Biochemistry and Molecular Biology
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Enolase is a uniquitous cytosolic glycolytic enzymes, which catalyze the conversion of phosphoenolpyruvate (PEP) with 2-phosphoglycerate (PGA) of the glycolysis. Recent studies show that enolase not just play the role of enzyme-catalyzed glycolysis and gluconeogenesis in the process, it is a multi-faceted, multi-functional protein. It can be used as an autoantigen involved in the immune response, also available as a c-myc promoter binding protein negatively regulates the expression of c-myc, involved in the regulation of cell growth, cell differentiation, also as plasminogen receptor involved in fibrinolysis processes. Antibodies (Abs) against a-enolase have been detected in a large variety of infectious and autoimmune diseases. These Abs might arise as a consquence of a microbial infection or uncontrolled growth or proliferation of cells in specific organs in pathophysiological conditions. This paper aims to generation monoclonal antibody of enolase and analysis of its epitope to explore the potential value of enolase antibody in physiological and pathology activity.In this paper, recombinant plasmid of human enolase gene were constructed and expressed successfully in Escherichia coli by molecular cloning technique, then the enolase of human and Escherichia coli immunized mice to generate antibodies. After cell fusion, positive clone selection and cloning culture, to generation monoclonal antibody against enolase. The results show as follows:1. The enolase gene of human was amplified by PCR, and then the recombinant expression plasmid pET28a-eno was gotten after restruction enzyme digestion. The expression plasmid was transformed into Escherichia coli BL21(DE3) and induced expression. The soluble, high-efficiency expressions were detected at 37℃,0.5mM IPTG, 6h after SDS-PAGE analysis.2. The enolase of human and Escherichia coli were purified to immunize mice. The titer in serum was above 1.5 in diluted a hundred fold which indciate the mice was successful immunization and can be used for cell fusion. After cell fusion in vivo and selection, one monoclonal antibody named 6B2 was successfully generated. mAb 6B2 was belongs to the IgGl subtype. The monoclonal antibody can react with both human enolase and Escherichia coli enolase in ELISA testing, while the reaction of 6B2 with human-specific enolase reaction was stronger than Escherichia coli enolase.3. The reaction of mAb 6B2 to enolase in vivo was verified by immunofluorescence and immunohistochemical techniques. Because of the high conservion in evolution, the mouse peritoneal macrophages were test for reaction of mAb 6B2 to enolase. The 6B2 antibody was used as first antibody to verify the specificity and localization in the cell. The results showed that mAb 6B2 binding with enolase localized in the cytoplasm and cell membrane in mouse peritoneal macrophages.4. The epitope of mAb 6B2 was screened by the phage display library. After three rounds of amplification and panning, the specific binding phage DNA was extracted and sequenced. The sequence analysis showed that the same sequence of the inserts was CGCACTGCCCAGTAGACCTGAGAACTTCAGTTCCAG, the corresponding amino acid sequence was DLDFKSPDDPSR, which is same to the amino acid to the 259-262 and 267-269 of human enolase. The amino acid sequence was just in the convex ring in the three-dimensional organization by Ellipro and PDB respectively.
Keywords/Search Tags:enolase, monoclonal antibody, phage display library
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