家蚕BmPLIN基因的克隆表达及定位研究

he cDNA library of pupae phase of silkworm has been constructed in our institute. According to the large-scale scanning of the library, a cDNA with 918 bp ORF was identified which encodes a perilipin protein. This gene encodes 305 amino acid residues with the predicted molecular weight of 32.497 kD and has been named BmPLIN by us. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli Rosetta and successfully expressed as inactive inclusion bodies. Following washing and purification, polyclonal antibodies was obtained against the recombinant protein in an immuned rabbit. The titer of the polyclonal antibodies measured by ELISA reaches more than 1:12800. In order to analysis the transcriptional and expressive level of BmPLIN gene using Real-time PCR and Western blotting, we extracted the mRNA and natural proteins in different tissues of the fifth instar larvae of Bombyx mori . From higher to lower, the amount of mRNA expressed in different tissues of the fifth instar larvaei is: gonad, fatty body, gut, epidermis, Malpighian tubule, head, silk glands, stigma. The result of Western blotting shows that in the gonad of the fifth instar larvae is BmPLIN protein-abundant. Moreover, Using fluorescent antibody and DAPI counter-stain for sub-cellular localization, we found the protein is both in cytoplasm and nuclei