家蚕小热休克蛋白Bmhsp21.4基因的研究

We clone a silkworm pupa cDNA sequnce and found that the cDNA sequence is known as sHSP of Bombyx mori, named BmHSP21.4 .The gene contained an ORF of 564 bp encoding a polypeptide of 187 amino acids with a predicted molecular mass of 21.4 kDa and a theoretical pI of 5.958. The ORF of Bmhsp21.4 gene was inserted into the pET-28a(+) vector and constructed the recombinant pET-28a-Bmhsp21.4, IPTG(1 mM) was added to the culture medium to induce the expression of recombinant protein. The molecular weight of this fusion protein was 24.862 Da. The induced cells were harvested and subjected to nickel affinity column chromatography to purify fusion protein. In order to obtain polyclonal antibody, the purified fusion protein was used to immunize a male New Zealand rabbit. Proteins from fatty body, testes, epidermis, trachea, ovary, malpighian tubule, silk glands, intestine, head, blood from the fifth instar larvae and different stages of silkworm and were extracted and analyzed by Western blotting and RT-PCR. The result showed that BmHSP21.4 existed in a lot of tissues in which head is low. We compared the detection of BmHSP21.4 in some posterior during the 5th instar of larvae with different heat-shock time. However, upon heat shock, BmHSP21.4 expression was significantly increased at 2.5-3h. We also compared the detection of BmHSP21.4 in some posterior during the 5th instar of larvae with different heat-shock temperature. We found BmHSP21.4 expression was significantly increased at 40℃. Finally, we used anti-BmHSP21.4 antibody and examined the effect of heat stress on localization of BmHSP21.4 by indirect immunofluorescence assays. BmHSP21.4 can be found in both the nucleus and cytoplasm but is primarily located in the nucleus.