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Cloning Expression of Selenoprotein W from Chicken and Characterization of Monoclonal Antibodies Against Their Recombinant Proteins

Author: XuMing
Tutor: XuShiWen
School: Northeast Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Chicken Selenoprotein W Monoclonal antibodies The prokaryotic expression Indirect ELISA
CLC: S831
Type: Master's thesis
Year: 2011
Downloads: 45
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Abstract


Selenium is an important trace elements necessary for human and animal life activities. Selenoprotein selenium covalently bound to the protein in a form of selenocysteine. Selenoprotein W (SelW) is a low molecular weight cell selenoproteins. In the different biological SelW the amino acid sequences are highly homologous. Previous studies have shown the SelW were expressed in a variety of tissues and organs, and a higher expression levels in skeletal muscle and brain. The current research mammals and rodents SelW function, SelW having regulating striated muscle metabolism, calcium metabolism and anti-oxidation effect. However, For chicken SelW the function has not been reported. For an in-depth study of the function of the chicken selenoprotein W cDNA under the chicken SelW of primers were designed, containing the complete ORF cDNA sequence was amplified by overlap extension PCR cDNA in Sec codon mutation Sequence analysis results with the reference sequence homology of more than 99%, and mutation products were cloned into the pGEX-6P-1 expression vector, the recombinant expression plasmid pGEX-SelW transformed into Rosetta host strain induced by IPTG, the recombinant fusion protein efficient expression, and are expressed mainly in the form of inclusion bodies, purified recombinant fusion protein by the method of plastic cutting, re-use to the thrombin cut off the GST tag, after the second cut gel purified target protein. Polyclonal antibody by Western blotting showed that the fusion protein with the corresponding the GST-SelW of reaction can produce specific bands, does not react with empty vector after induction of GST-tagged proteins. Purified protein BABL / c mice, cell fusion technology, indirect ELISA screening and cell subclones, two hybridoma, named 1B8 and 4H5. Identified by the monoclonal antibody subclasses kit, were IgM class antibody supernatant titers were 1:6400. Prepared 1B8 and 4H5 ascites monoclonal antibody, ascites titer of 1:12800 and 1:6400. 2 hybridoma chromosome number of 103 ± 5, SP2 / 0 myeloma cells significantly more than 65 to 75. 2 continuous passage 3 months and frozen after recovery did not affect the stability of the antibody-secreting hybridoma cells in vitro. No cross reaction with the GST labels and chicken selenoprotein N. Affinity analysis showed that 2 monoclonal antibody affinity target protein is higher than the recombinant fusion protein and chicken SelW of synthetic peptide and high affinity monoclonal antibody sensitive, stable and fast detection applications, these provide the necessary guarantees. In short, the success of two monoclonal antibodies prepared for the chicken SelW rapid, specific detection detection reagents, and also provide the material basis for the chicken SelW function further research.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Poultry > Chicken
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