Study On Expression Characteristics Of BmCycH Gene In Silkworm, Bombyx Mori

The catalytic subunit of the metazoan Cdk-activating kinase(CAK) is cyclin-dependent kinase 7(Cdk7). Activation of Cdk7 requires its association with a regulatory subunit, cyclinH. In mammalian cells, Cdk7, cyclin H and MAT1, function as the CAK which phosphorylates the conserved threonine residue within the T loop of several Cdks. However, most researches of cyclin H focused on vertebrate, seldom were done on invertebrate.The full open reading frame (ORF) of Bombyx mori cyclin H was obtained from silkworm pupa by RT-PCR and named BmCycH (Bombyx mori cyclin H), of which the accession number was AV406047. After cloned into pET-28a(+) and expressed in E.coli BL21(DE3), the BmCycH was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 32.0 kD, characterized by SDS-PAGE. This result proved that His-tag BmCycH was expressed in the right form. After being purified with Ni2+ affinity chromatography the recombinant protein was as antigen against which the antibody was raised by immune rabbit. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA.We also compared the quantity of BmCycH mRNA in different stages of the fifth instar larva and different tissues of silkworm. The expression level in the moth was highest, but in the egg was the lowest; and in the different tissues, the expression level in the head was the highest and the silk gland was the lowest. Western blot analysis revealed that the protein was expressed in all silkworm tissues and 4 developmental phases examined, with highest expression in the moth, pupa and larvae, lowest in egg. It may suggest that BmCycH plays a role in the differentiation and development of silkworm. Using fluorescent antibody for subcellular localization, we preliminarily localized the protein in the nucleus in BmN.