Study On Heat Shock Protein21.4 Gene Of Bombyx Mori

sHSPs are stress-inducible molecular chaperones that range in size from 12 to 43 kD and include theα-crystallin domain consisting of 80-100 amino acids . According to its molecular weight size and the function, it was divided into three families generally: HSP70, HSP90 and small molecular heat shock protein family.The small molecular heat shock protein is an extremely rich multiple family, the different kinds of organism have the different types, the molecular weight in 15-30 kD.We clone a silkworm pupa cDNA sequnce and found that the cDNA sequence is known as sHSP of Bombyx mori which name is 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 kD 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 kD, characterized by mass- spectrum after being further purified on 10 RI FPLC.We 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 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 2.5-3 h. 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℃-45℃. 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 could be found in both the nucleus and cytoplasm but was primarily located in the nucleus.