Analysis Of The Expression And Function Of Small Heat Shock Protein 22.6 In Bombyx Mori

Small heat shock proteins (sHSP) are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stress. As a molecular chaperone, sHSPs have been shown to be associated with a number of biological processes including cell proliferation and differentiation.We obtained a sequence from the cDNA library constructed by our laboratory, which has an ORF of 597 bp coding 198 amino acid residues of the analysis of homology. The protein is a highly conservative silkworm small heat shock protein 22.6(BmHSP22.6) with a conservedα-crystallin domain. In order to obtain the polyclonal antibodies of BmHSP22.6, the ORF was inserted into the pET-28a vector and constructed the recombinant pET-28a-BmHSP22.6. The fusion protein was expressed successfully in E.coli BL21 Star(DE3) by adding IPTG (1mM) for induction, and it was soluble. The soluble His-tag BmHSP22.6 protein was purified via nickel affinity column chromatography and used to immunize a male New Zealand rabbit. The polyclonal antibody whose titer was larger than 1: 6400 was obtained, and its specific reaction was well detected in Western blot analysis. It was also compared that the quantity of BmHSP22.6 in different tissues of the fifth instar larva and different stages of silkworm. The result showed that BmHSP22.6 existed in adult and a lot of tissues in which epidermis and head were higher. BmHSP22.6 expression was significantly increased by heat shock the spinning larva and pupa detected by ELISA and Western blot analysis. Anti-BmHSP22.6 antibody was used to examine the effect of heat stress on localization of BmHSP22.6 in indirect immunofluorescence assays. BmHSP22.6 was found in cytoplasm, after heat treated, the expression level of BmHSP22.6 became higher. These results provided a better understanding of cellular protection mechanisms against environmental stress such as heat shock. Finally, it was strongly indicated that BmHSP22.6 was of chaperone-like activity in that the BmHSP22.6 protein effected aggregation of lysozyme on concentration base.