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Analysis of the Expression and Function of Small Heat Shock Protein 22.6 in Bombyx Mori

Author: ZhengBi
Tutor: ZhangYaoZhou
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: The silkworm small heat shock protein 22.6 Prokaryotic expression Polyclonal antibody Heat shock Molecular chaperone
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 37
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Abstract


Small heat shock proteins (small heat shock protein, sHSP) family of proteins is induced by heat treatment . As an important molecular chaperone , heat shock protein involved in cytoskeletal stability, cell migration , regulate cell growth and differentiation , and the anti-apoptotic cells . Silkworm cDNA library constructed in our laboratory , we obtained a sequence of a total length of 841 bp open reading frame (ORF) of 597 bp in length , encoding 198 amino acid residues , contains a highly conserved alpha - crystallin structure domains. In order to prepare anti - BmHSP22.6 protein polyclonal antibody , we will BmHSP22.6 gene was cloned into the expression vector pET - 28a provided on the fusion protein expression plasmid pET - 28a - BmHSP22.6 successfully constructed . The recombinant was transformed into E. coli BL21 Star ( DE3 ) , a final concentration of 1 mM IPTG - induced recombinant bacteria successfully expressed fusion protein His - BmHSP22.6 , sonication found that the protein in soluble form . By the the affinity chromatography target protein His - BmHSP22.6 as antigen male New Zealand rabbits , prepared the polyclonal antibody , the indirect ELISA measured antibody titer of 1:6400 , Western blot detection of antibodies specific good , the antibody correspond to the requirements for further experiments . Western blot analysis found that the highest expression of BmHSP22.6 protein at home saturniid of period and the head of the fifth instar larvae , epidermis . Silking stage larvae and pupae of silkworm heat shock experiment , to extract different organizations protein , ELISA and semi - quantitative method and detected by Western blot after heat shock treatment of different organizations protein expression changes . Immunofluorescence The experimental results show that , BmHSP22.6 protein exists in silkworm ovary cells the BmN the cytoplasm , after heat shock treatment , the expression level increased significantly . These provide a good explanation of the small heat shock protein protection mechanisms of resistance to environmental stress ( such as thermal stimulation ) . The research BmHSP22.6 on lysozyme aggregation , different concentrations BmHSP22.6 found in varying degrees, inhibition of lysozyme aggregation , having a molecular chaperone function .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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